Characterization and correction of dystrophic iPS cells

To address the feasibility of using disease-specific iPS cells and genetic correction in the context of Duchenne muscular dystrophy (DMD), we utilized the dystrophin/utrophin dKO mouse model as the source of tail-tip fibroblasts (TTFs), as well as recipients of iPS-derived therapeutic myogenic cell preparations (Fig. 1a). This choice was based on the fact that mdx mice, although a favourite model for DMD15, present a mild phenotype, attributed to compensatory overexpression of the dystrophin-related protein, utrophin16. The dKO mice, which lack both dystrophin and utrophin, present a severe phenotype characterized by progressive muscle wasting, impaired mobility, abnormal breathing pattern, cardiomyopathy and premature death17,18, which more closely resembles DMD in human patients. To restore the dystrophin-glycoprotein complex (DGC), we chose to re-express a μUTRN transgene, which has been shown to ameliorate the dystrophic phenotype19,20,21,22. This approach tests a vector that would potentially be preferred in human patients because it would avoid the immune response that is elicited by the dystrophin-naive immune system (the mouse model tests function of μUTRN in this context, but not immune aspects). As summarized in Fig.1a, the therapeutic strategy applied in the present study involved (1) reprogramming of dystrophic donor fibroblasts into iPS clones, (2) genetic repair of selected iPS clones with μUTRN using the non-viral Sleeping Beauty Transposon (Tn) System, (3) in vitro differentiation of corrected iPS cell clones into myogenic progenitors and (4) transplantation of corrected myogenic precursors into dystrophic dKO mice (mdx; utrn−/−).

Figure 1: Ex vivo correction of dystrophic iPS cells. (a) Scheme of the ex vivo gene therapy approach, which involves (1) reprogramming of dystrophic donor-derived fibroblasts into iPS cells, (2) genetic repair of iPS cells with the μUTRN transgene using the Sleeping Beauty Tn system, (3) generation of myogenic progenitors from corrected iPS cells through Pax3 induction and (4) transplantation of corrected myogenic precursors into dystrophic donor mice. (b) The Sleeping Beauty Tn system: the Tn contains the hEF1α-eIF4g promoter, the μUTRN gene and an iresGFP. The whole transgene is flanked by the terminal inverted repeats (IR/DR, arrowheads), each containing two binding sites for the transposase (DR, yellow arrows). The transposase protein SB100X (red spheres) catalyses integration of the Tn into the genome with high efficiency. (c) Left panel: Graphic bars represent percentage of GFP+ cells obtained before each sorting. Green bars correspond to iPS cells nucleofected with pKT2/μUTRN-iresGFP/SB100X, whereas yellow bars represent controls, iPS cells transduced with pKT2/μUTRN-IresGFP only (no transposase). Data are mean±s.e.m. of three independent samples. (d) FACS profile for GFP shows stable expression of μUTRN in corrected iPS cells (right panel), whereas control iPS cells are GFP- (left panel). (e) Flow-cytometric analyses for Flk-1 and PDGFαR expression on day 5 EBs of Pax3-induced uncorrected (upper panel) and corrected iPS (lower panel) cells. The PDGFαR+Flk-1− cell fraction was gated (red square on left panels) and analysed for the expression of GFP, representing μUTRN+ cells (middle panels), and mCherry, representing Pax3+ cells (right panels). (f) Phase-contrast images of monolayers under proliferation (left) and differentiation (right) culture conditions. (g) Immunofluorescence staining for μUTRN (green) in proliferating iPS-derived myogenic progenitors (left) and their derivative myotubes (right). Cells are co-stained with 4,6-diamidino-2-phenylindole (DAPI) (blue). Scale bar, 200 μm. (h) Quantitative PCR analyses indicate relative expression of μUTRN in corrected iPS-derived myogenic cells under proliferation (P) and differentiation (D) conditions. Actin was used as house-keeping gene. UNC: uncorrected cells. Error bars represent s.e.m. from three replicates of three independent experiments. (i) Immunoblotting with anti-UTRN and anti-FLAG antibodies confirms the presence of 163 kDa μUTRN-only in corrected cells. Control uncorrected cells are labeled as C. Full size image

Dystrophic iPS cells were generated by retroviral transduction of TTF cells using Oct4, Klf-4 and Sox2 (ref. 23). iPS clones were initially screened by morphology, SSEA-1 expression and alkaline phosphatase activity (Supplementary Fig. S1a,b). Based on these markers, a subset of clones was selected for additional analysis, which included immunofluorescence staining to pluripotency markers (Supplementary Fig. S1c), as well as assessment of their ability to form embryonic mesoderm following in vitro differentiation into embryoid bodies (EBs), as evidenced by Flk-1 and PDGFαR expression (Supplementary Fig. S1d–e). After this analysis, one iPS clone (C3) was chosen for the studies presented here. These cells displayed normal karyotype and exhibited the ability to develop typical teratomas (Supplementary Fig. S1f,g). Importantly, following EB differentiation, iPS cells showed downregulation of pluripotency markers and normal expression of imprinted genes (Supplementary Fig. S1h–i, respectively), which has been previously shown to distinguish fully reprogrammed iPS cells24.

Dystrophic iPS cells were then corrected with the μUTRN transgene (R4-R21). This gene is a contracted version of UTRN-lacking sequences encoding spectrin-like repeats 4 through 21, but containing the N-terminal domain that binds to F-actin, and the C-terminal domain (C/C cysteine-rich domain) that interacts with the dystrophin–glycoprotein complex. This gene complements both the loss of dystrophin and utrophin20. The Sleeping Beauty Tn system has several advantages including efficient gene transfer and stable gene expression in mouse and human ES cells25,26, and reduced likelihood of insertional mutagenesis27. We developed a T2-inverted terminal repeat Tn vector (pKt2/μUTRN) carrying a 7.3-kb engineered transgene containing μUTRN and an iresGFP reporter (Fig. 1b), which allows for FACS selection of μUTRN-corrected iPS cells. SB100X (Fig. 1b) is a recently engineered hyperactive variant that yields high levels of Tn integration, leading to efficient and stable gene transfer28. We observed a stable gene-transfer frequency of 0.3%, 1 week after nucleofection with Tn and transposase vectors (Fig. 1c). Green fluorescent protein (GFP+) cells were sorted, expanded and re-sorted, giving rise to a homogeneous and stable population of μUTRN-corrected iPS cells (>90% GFP+) (Fig. 1c, right panel). The expression of μUTRN was confirmed by quantitative PCR (Supplementary Fig. S2a). Corrected iPS cells maintained a normal karyotype (Supplementary Fig. S2b).

Skeletal myogenic progenitors from gene-corrected iPS cells

To enable the efficient generation of skeletal myogenic progenitors, both μUTRN-corrected and control (uncorrected) iPS cells (Fig. 1d) were modified to allow doxycycline-regulated conditional Pax3 expression, which we have previously shown allows the derivation of repopulating myogenic progenitors from ES cells12 (Supplementary Fig. S3a). Pax3+ cells (mCherry+) were detected only when dox was added to the culture medium (Supplementary Fig. S3b). This has been confirmed by western blot analysis (Supplementary Fig. S3c). To isolate skeletal myogenic precursors, Pax3 was induced from day 3 to day 5 of EB differentiation, at which point paraxial mesoderm progenitors were isolated based on the expression of PDGFαR and lack of Flk-1 (Fig. 1e, left panels)12. In the case of corrected iPS-derived EBs, cells were further purified based on positivity for GFP, which is co-expressed with μUTRN (Fig. 1e, lower middle panel). Because Pax3 is co-expressed with mCherry, both control and corrected dox-induced EBs were mCherry+ (Fig. 1e, right panels), whereas non-induced control counterparts were mCherry− (Supplementary Fig. S3d).

PDGFαR+Flk-1−GFP+-sorted cell preparations from corrected iPS-derived EBs (Fig. 1e) were expanded in the presence of basic fibroblast growth factor (bFGF) and doxycycline. Proliferating myogenic precursors emerged from these cultures (Fig. 1f, left panels). When differentiation was induced in vitro, these had the ability to undergo final maturation, giving rise to multinucleated myotubes (Fig. 1f, right panels) that exhibited elevated fusion index (82±8%; Supplementary Fig. S3e) (details in methods). Importantly, abundant and stable expression of μUTRN was observed in corrected myogenic progenitors and their derivative myotubes, as shown by immunofluorescence staining (Fig. 1g), quantitative PCR (Fig. 1h) and western blot analysis (Fig. 1i). No utrophin was detected in control uncorrected myogenic preparations (Fig. 1i).

As anticipated, proliferating myogenic progenitors were characterized by abundant expression of Pax3 as well as Myf5 (Supplementary Fig. S4a). Under these conditions, MyoD was observed in fewer nuclei, whereas terminal differentiation markers, such as MyHC, were barely detected (Supplementary Fig. S4a). This profile changed upon induction of myogenic differentiation, as multinucleated myotubes expressed high levels of MyHC and MyoD, and displayed significant downregulation of Pax3 and Myf5 (Supplementary Fig. S4b). These results were further confirmed by real-time RT–PCR (Supplementary Fig. S5). Flow-cytometric analysis of μUTRN-corrected myogenic progenitors showed homogeneous expression of M-cadherin (99%), CD56 (82%), VCAM1 (85%), SYND-4 (98%) and CXCR4 (98%) (Supplementary Fig. S4c), surface markers characteristic of satellite and myogenic progenitor cells29.

Regenerative potential of gene-corrected myogenic cells

To assess whether these corrected autologous cells would engraft and differentiate into muscle in vivo, μUTRN-corrected iPS-derived myogenic precursors were transplanted into the left tibialis anterior (TA) muscles of 3-week-old dKO mice, whereas the contra-lateral TA (right) was injected with PBS. Because the dKO strain presents a much more severe phenotype, we did not pre-injure with cardiotoxin (CTX) to enhance engraftment, as previously described for transplantations in mdx mice12. Mice received immunosuppression daily (Tacrolimus) as both dystrophin and utrophin are foreign proteins to these mice, in addition to GFP. Three weeks following transplantation, cryosections of TA muscles were evaluated for engraftment by immunofluorescence staining for utrophin using a polyclonal antibody that specifically recognizes the amino-terminal epitope preserved in the μUTRN transgene. As expected17,18, no expression of utrophin was detected in PBS-injected control muscles of the contra-lateral leg (Fig. 2a). On the contrary, TA muscles that had been transplanted with μUTRN-corrected iPS-derived myogenic precursors demonstrated substantial engraftment, as showed by the clear expression of utrophin in recipient muscles (Fig. 2b). Quantification of μUTRN+ myofibers showed engraftment average levels of 19±7.6% in transplanted mice (Fig. 2c). Analyses for β-dystroglycan, α1-syntrophin and neuronal nitric oxide synthase in consecutive sections revealed contiguous expression of these proteins across long sections of engrafted μUTRN+ myofibers (Fig. 2b). These data show that μUTRN-corrected iPS-derived myogenic precursors are able to engraft in vivo, and, more importantly, μUTRN expression was able to restore other components of the DGC, which are missing in the absence of dystrophin (Fig. 2a)17,18,30,31. Another critical aspect is that no tumour was detected in any of the transplanted mice (total of 20 dKO mice) or in NOD/SCID mice injected with these cell preparations up to 3 months post transplantation. We have previously shown that by incorporating positive selection for paraxial mesoderm markers, teratoma-forming cells can be eliminated from mouse ES-derived cell preparations12.

Figure 2: Engraftment of μUTRN-corrected iPS-derived myogenic precursors in dKO mice. Serial cross-sections (total length of 1000 μm) of TA muscles collected from dKO mice that had been injected with PBS (contra-lateral leg) (a) or myogenic progenitors (b) were stained with antibodies to utrophin and several members of the dystrophin protein complex, including β-dystroglycan (β-DG), α1-syntrophin (α1-SYN) and neuronal nitric oxide synthase (nNOS) (red). These proteins were detected only in muscles transplanted with μUTRN-corrected myogenic precursors, in which they were found expressed in a contiguous manner across long sections of muscle fibre, as shown by the asterisks. Roman numbers within these images indicate the order of serial sections. PBS-injected dKO mice showed no signal for any of these proteins. 4,6-Diamidino-2-phenylindole (DAPI) is shown in blue. Scale bar, 50 μm. (c) Quantification of μUTRN+myofibers in these engrafted muscles (n=8). For each muscle, 3–4 representative cross-sections at 2-mm intervals were counted. For PBS control groups, we examined 20 random sections. Error bars represent s.d. Full size image

Engrafted corrected cells reestablish functional properties

To investigate whether engraftment was accompanied by improvement in muscle function, we evaluated in a blinded manner the contractile properties of transplanted muscles compared with their contra-lateral leg controls. Engrafted muscles showed markedly superior isometric tetanic force (Fig. 3a) and increased absolute and specific force (Fig. 3b, respectively) when compared with their respective contra-lateral PBS-injected TA muscles. No changes were observed in cross-sectional area (CSA) (Supplementary Fig. S6c) or weight (Supplementary Fig. S6d), as previously observed for ES-derived myogenic progenitors using this delivery route12. Data from the fatigue test showed no differences between transplanted and control groups (Fig. 3d), suggesting that levels of engraftment were not sufficient to restore this parameter.

Figure 3: Functional improvement and satellite cell engraftment of transplanted muscles. (a) Representative examples of isometric force tracking in TA muscles that had been injected with corrected cells or PBS (contra-lateral leg) (green and grey lines, respectively). For reference, untreated dKO and wild-type Bl6 mice controls are shown (dashed and black line, respectively). (b,c) Effect of cell transplantation on absolute (F0) and specific (sF0=F0 normalized to CSA) force, respectively. Error bars represent s.e.m. from a total of 13 transplanted mice. *P<0.05, **P<0.01 and ***P<0.001. (d) Fatigue index: time for force to decline to 30% of maximal value during continuous stimulation of muscle at 150 Hz. (e) In situ localization images show the presence of donor-derived satellite cells in engrafted dKO mice, as evidenced by the presence of Pax7+ (red) and GFP+ (green) cells under the basal lamina (indicated by arrows). Arrowheads denote endogenous Pax7+ GFP− satellite cells. (f) Engrafted cells respond to CTX injury, giving rise to new myofibers as evidenced by the co-expression of μUTRN (red) and embryonic MHC (green). White arrows indicate μUTRN+/eMHC+ donor-derived newly formed myofibers, and arrowheads point to μUTRN−/eMHC+ host-derived newly formed myofibers. Scale bar, 50 μm. DAPI, 4,6-diamidino-2-phenylindole. Full size image

Transplanted cells seed the satellite cell compartment

Next, we examined whether μUTRN-corrected myogenic precursors have the ability to seed the satellite cell compartment, and therefore to respond to injury (Supplementary Fig. S6d). To facilitate the detection of donor-derived satellite cells, corrected myogenic precursors were labelled with a lentiviral vector encoding GFP (Supplementary Fig. S6d). Three weeks following transplantation, we could clearly identify the presence of Pax7+GFP+ cells beneath the basal lamina (Fig. 3e), indicative of donor-derived satellite cells. This was confirmed by another experimental cohort, in which we promoted CTX injury in dKO mice that had been transplanted 3 weeks prior with μUTRN-corrected myogenic precursors (not labelled with GFP) (Supplementary Fig. S6d). One week after injury, we detected the presence of donor-derived newly formed corrected myofibers, as shown by the presence of μUTRN+/embryonic MHC+ myofibers (Fig. 3f). Recipient-derived newly formed myofibers (μUTRN−/embryonic MHC+) were found in transplanted mice (Fig. 3f) as well as in PBS-injected controls (contra-lateral leg; Supplementary Fig. S6e), as expected. The donor-derived newly formed myotubes (eMHC+μUTRN+; Fig. 3f) could be derived from iPS-derived satellite cells (most likely because there are donor-derived satellite cells in engrafted muscles; Fig. 3e), but could also form from a non-satellite myogenic precursor population that did not seed the satellite cell compartment and did not differentiate into myofibers. These results are of particular interest, as activation of corrected satellite cells could provide new μUTRN+ myofibers during the progress of the muscular dystrophy, slowing the progression of the disease.

Engrafted myofibers exhibit synaptic connections

To address whether engrafted μUTRN+ myofibers exhibited direct connection with motoneurons, we stained muscle sections with α-bungarotoxin (α-BTX). Our results clearly show the presence of nicotinic acetylcholine receptors at the neuromuscular junction of engrafted fibres (Fig. 4). In wild-type mice, as expected, utrophin was detected solely at the neuromuscular junction, along with α-BTX (Fig. 4c), whereas in PBS-injected controls, only α-BTX was present (Fig. 4b). All together, these data suggest that μUTRN-corrected iPS-derived myogenic precursors integrate with the neuromuscular system.

Figure 4: Engrafted μUTRN+ myofibers exhibit direct synaptic connections to motoneurons. Representative images show AChRs labelled with α-BTX in TA muscles that had been injected with corrected iPS-derived myogenic progenitors (a) or PBS (b). TA muscles from wild-type (WT) mice are also shown as a reference (c). Utrophin is indicated in red and α-BTX is shown in green. 4,6-Diamidino-2-phenylindole (DAPI) is shown in blue. Scale bar, 50 μm. Full size image

Systemic cell transplantation in dKO mice

Because reaching disparate muscle groups is critical when considering clinical application of cell therapy for DMD, we assessed engraftment levels following the systemic delivery of μUTRN-corrected iPS-derived myogenic precursors. Intravenous injection of these cell preparations resulted in distribution of donor-derived myofibers in several muscles, including TA, gastrocnemius lateralis and peroneal (Fig. 5a–c; Supplementary Table S1), of all the five mice that had been transplanted. In one of these mice, we were able to detected μUTRN-donor-derived myofibers in the diaphragm (Supplementary Fig. S7a). No utrophin signal was detected in any muscles from the PBS-injected mice (Supplementary Figs S7b and S8; Supplementary Table S1). In this cohort of transplanted mice, no engraftment was observed in non-skeletal muscle tissues including heart, lung and liver (Supplementary Fig. S9). This was also the case following the systemic injection of mouse ES cells, as previously reported12.