Heparan Sulfate Chain-Conjugated Laminin-E8 Fragments Advance Paraxial Mesodermal Differentiation Followed by High Myogenic Induction from hiPSCs.

Human-induced pluripotent stem cells (hiPSCs) have great therapeutic potential. The cell source differentiated from hiPSCs requires xeno-free and robust methods for lineage-specific differentiation. Here, a system is described for differentiating hiPSCs on new generation laminin fragments (NGLFs), a recombinant form of a laminin E8 fragment conjugated to the heparan sulfate chains (HS) attachment domain of perlecan. Using NGLFs, hiPSCs are highly promoted to direct differentiation into a paraxial mesoderm state with high-efficiency muscle lineage generation. HS conjugation to the C-terminus of Laminin E8 fragments brings fibroblast growth factors (FGFs) bound to the HS close to the cell surface of hiPSCs, thereby facilitating stronger FGF signaling pathways stimulation and initiating HOX gene expression, which triggers the paraxial mesoderm differentiation of hiPSCs. This highly efficient differentiation system can provide a roadmap for paraxial mesoderm development and an infinite source of myocytes and muscle stem cells for disease modeling and regenerative medicine.

Dual CRISPR-Cas3 system for inducing multi-exon skipping in DMD patient-derived iPSCs.

To restore dystrophin protein in various mutation patterns of Duchenne muscular dystrophy (DMD), the multi-exon skipping (MES) approach has been investigated. However, only limited techniques are available to induce a large deletion to cover the target exons spread over several hundred kilobases. Here, we utilized the CRISPR-Cas3 system for MES induction and showed that dual crRNAs could induce a large deletion at the dystrophin exon 45-55 region (∼340 kb), which can be applied to various types of DMD patients. We developed a two-color SSA-based reporter system for Cas3 to enrich the genome-edited cell population and demonstrated that MES induction restored dystrophin protein in DMD-iPSCs with three distinct mutations. Whole-genome sequencing and distance analysis detected no significant off-target deletion near the putative crRNA binding sites. Altogether, dual CRISPR-Cas3 is a promising tool to induce a gigantic genomic deletion and restore dystrophin protein via MES induction.

Establishment of quantitative and consistent in vitro skeletal muscle pathological models of myotonic dystrophy type 1 using patient-derived iPSCs.

Myotonic dystrophy type 1 (DM1) is caused by expanded CTG repeats (CTGexp) in the dystrophia myotonica protein kinase (DMPK) gene, and the transcription products, expanded CUG repeats, sequester muscleblind like splicing regulator 1 (MBNL1), resulting in the nuclear MBNL1 aggregation in the DM1 cells. Loss of MBNL1 function is the pivotal mechanism underlying the pathogenesis of DM1. To develop therapeutics for DM1, proper human in vitro models based on the pathologic mechanism of DM1 are required. In this study, we established robust in vitro skeletal muscle cell models of DM1 with patient-derived induced pluripotent stem cells (iPSCs) using the MyoD1-induced system and iPSCs-derived muscle stem cell (iMuSC) differentiation system. Our newly established DM1 models enable simple quantitative evaluation of nuclear MBNL1 aggregation and the downstream splicing defects. Quantitative analyses using the MyoD1-induced myotubes showed that CTGexp-deleted DM1 skeletal myotubes exhibited a reversal of MBNL1-related pathologies, and antisense oligonucleotide treatment recovered these disease phenotypes in the DM1-iPSCs-derived myotubes. Furthermore, iMuSC-derived myotubes exhibited higher maturity than the MyoD1-induced myotubes, which enabled us to recapitulate the SERCA1 splicing defect in the DM1-iMuSC-derived myotubes. Our quantitative and reproducible in vitro models for DM1 established using human iPSCs are promising for drug discovery against DM1.

Single-cell RNA-seq reveals heterogeneity in hiPSC-derived muscle progenitors and E2F family as a key regulator of proliferation.

Human pluripotent stem cell-derived muscle progenitor cells (hiPSC-MuPCs) resemble fetal-stage muscle progenitor cells and possess in vivo regeneration capacity. However, the heterogeneity of hiPSC-MuPCs is unknown, which could impact the regenerative potential of these cells. Here, we established an hiPSC-MuPC atlas by performing single-cell RNA sequencing of hiPSC-MuPC cultures. Bioinformatic analysis revealed four cell clusters for hiPSC-MuPCs: myocytes, committed, cycling, and noncycling progenitors Using FGFR4 as a marker for noncycling progenitors and cycling cells and CD36 as a marker for committed and myocyte cells, we found that FGFR4+ cells possess a higher regenerative capacity than CD36+ cells. We also identified the family of E2F transcription factors are key regulators of hiPSC-MuPC proliferation. Our study provides insights on the purification of hiPSC-MuPCs with higher regenerative potential and increases the understanding of the transcriptional regulation of hiPSC-MuPCs.

Collagen-VI supplementation by cell transplantation improves muscle regeneration in Ullrich congenital muscular dystrophy model mice.

BACKGROUND: Mesenchymal stromal cells (MSCs) function as supportive cells on skeletal muscle homeostasis through several secretory factors including type 6 collagen (COL6). Several mutations of COL6A1, 2, and 3 genes cause Ullrich congenital muscular dystrophy (UCMD). Skeletal muscle regeneration deficiency has been reported as a characteristic phenotype in muscle biopsy samples of human UCMD patients and UCMD model mice. However, little is known about the COL6-dependent mechanism for the occurrence and progression of the deficiency. The purpose of this study was to clarify the pathological mechanism of UCMD by supplementing COL6 through cell transplantation. METHODS: To test whether COL6 supplementation has a therapeutic effect for UCMD, in vivo and in vitro experiments were conducted using four types of MSCs: (1) healthy donors derived-primary MSCs (pMSCs), (2) MSCs derived from healthy donor induced pluripotent stem cell (iMSCs), (3) COL6-knockout iMSCs (COL6KO-iMSCs), and (4) UCMD patient-derived iMSCs (UCMD-iMSCs). RESULTS: All four MSC types could engraft for at least 12 weeks when transplanted into the tibialis anterior muscles of immunodeficient UCMD model (Col6a1KO) mice. COL6 protein was restored by the MSC transplantation if the MSCs were not COL6-deficient (types 1 and 2). Moreover, muscle regeneration and maturation in Col6a1KO mice were promoted with the transplantation of the COL6-producing MSCs only in the region supplemented with COL6. Skeletal muscle satellite cells derived from UCMD model mice (Col6a1KO-MuSCs) co-cultured with type 1 or 2 MSCs showed improved proliferation, differentiation, and maturation, whereas those co-cultured with type 3 or 4 MSCs did not. CONCLUSIONS: These findings indicate that COL6 supplementation improves muscle regeneration and maturation in UCMD model mice.

Characterization of hiPSC-Derived Muscle Progenitors Reveals Distinctive Markers for Myogenic Cell Purification Toward Cell Therapy.

The transplantation of muscle progenitor cells (MuPCs) differentiated from human induced pluripotent stem cells (hiPSCs) is a promising approach for treating skeletal muscle diseases such as Duchenne muscular dystrophy (DMD). However, proper purification of the MuPCs before transplantation is essential for clinical application. Here, by using MYF5 hiPSC reporter lines, we identified two markers for myogenic cell purification: CDH13, which purified most of the myogenic cells, and FGFR4, which purified a subset of MuPCs. Cells purified with each of the markers showed high efficiency for regeneration after transplantation and contributed to the restoration of dystrophin expression in DMD-immunodeficient model mice. Moreover, we found that MYF5 regulates CDH13 expression by binding to the promoter regions. These findings suggest that FGFR4 and CDH13 are strong candidates for the purification of hiPSC-derived MuPCs for therapeutical application.

Generation of a transgene-free iPSC line and genetically modified line from a facioscapulohumeral muscular dystrophy type 2 (FSHD2) patient with SMCHD1 p.Lys607Ter mutation.

Facioscapulohumeral muscular dystrophy type2 (FSHD2), which constitutes approximately 5% of total FSHD cases and develops the same symptoms as FSHD type 1 (FSHD1), is caused by various mutations in genes including SMCHD1. We report the generation and characterization of an iPSC line derived from an FSHD2 patient carrying the SMCHD1 p.Lys607Ter mutation and its gene-corrected iPSC line which are free from transgene. These iPSC lines maintained normal karyotype, presented typical morphology, expressed endogenous pluripotency markers, and could be differentiated into ectodermal, mesodermal and endodermal cells, confirming their pluripotency.

A Liver Model of Infantile-Onset Pompe Disease Using Patient-Specific Induced Pluripotent Stem Cells.

Infantile-onset Pompe disease (IOPD) is a life-threatening multi-organ disease caused by an inborn defect of lysosomal acid α-glucosidase (GAA), which can degrade glycogen into glucose. Lack of GAA causes abnormal accumulation of glycogen in the lysosomes, particularly in the skeletal muscle, liver, and heart. Enzyme replacement therapy (ERT) with recombinant human GAA (rhGAA) is the only available treatment; however, its effect varies by organ. Thus, to fully understand the pathomechanism of IOPD, organ-specific disease models are necessary. We previously generated induced pluripotent stem cells (iPSCs) from three unrelated patients with IOPD and establish a skeletal muscle model of IOPD. Here, we used the same iPSC lines as the previous study and differentiated them into hepatocytes. As a result, hepatocytes differentiated from iPSC of IOPD patients showed abnormal accumulation of lysosomal glycogen, the hallmark of Pompe disease. Using this model, we also demonstrated that glycogen accumulation was dose-dependently restored by rhGAA treatment. In conclusion, we have successfully established an in vitro liver model of IOPD using patient-specific iPSCs. This model can be a platform to elucidate the underlying disease mechanism or to be applied to drug-screening. Moreover, our study also suggest that an iPSC-based approach is suitable for modeling of diseases that affect multiple organs like Pompe disease.

A patient-derived iPSC model revealed oxidative stress increases facioscapulohumeral muscular dystrophy-causative DUX4.

Double homeobox 4 (DUX4), the causative gene of facioscapulohumeral muscular dystrophy (FSHD), is ectopically expressed in the skeletal muscle cells of FSHD patients because of chromatin relaxation at 4q35. The diminished heterochromatic state at 4q35 is caused by either large genome contractions [FSHD type 1 (FSHD1)] or mutations in genes encoding chromatin regulators, such as SMCHD1 [FSHD type 2 (FSHD2)]. However, the mechanism by which DUX4 expression is regulated remains largely unknown. Here, using a myocyte model developed from patient-derived induced pluripotent stem cells, we determined that DUX4 expression was increased by oxidative stress (OS), a common environmental stress in skeletal muscle, in both FSHD1 and FSHD2 myocytes. We generated FSHD2-derived isogenic control clones with SMCHD1 mutation corrected by clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR associated 9 (Cas9) and homologous recombination and found in the myocytes obtained from these clones that DUX4 basal expression and the OS-induced upregulation were markedly suppressed due to an increase in the heterochromatic state at 4q35. We further found that DNA damage response (DDR) was involved in OS-induced DUX4 increase and identified ataxia-telangiectasia mutated, a DDR regulator, as a mediator of this effect. Our results suggest that the relaxed chromatin state in FSHD muscle cells permits aberrant access of OS-induced DDR signaling, thus increasing DUX4 expression. These results suggest OS could represent an environmental risk factor that promotes FSHD progression.

A Skeletal Muscle Model of Infantile-onset Pompe Disease with Patient-specific iPS Cells.

Pompe disease is caused by an inborn defect of lysosomal acid α-glucosidase (GAA) and is characterized by lysosomal glycogen accumulation primarily in the skeletal muscle and heart. Patients with the severe type of the disease, infantile-onset Pompe disease (IOPD), show generalized muscle weakness and heart failure in early infancy. They cannot survive over two years. Enzyme replacement therapy with recombinant human GAA (rhGAA) improves the survival rate, but its effect on skeletal muscle is insufficient compared to other organs. Moreover, the patho-mechanism of skeletal muscle damage in IOPD is still unclear. Here we generated induced pluripotent stem cells (iPSCs) from patients with IOPD and differentiated them into myocytes. Differentiated myocytes showed lysosomal glycogen accumulation, which was dose-dependently rescued by rhGAA. We further demonstrated that mammalian/mechanistic target of rapamycin complex 1 (mTORC1) activity was impaired in IOPD iPSC-derived myocytes. Comprehensive metabolomic and transcriptomic analyses suggested the disturbance of mTORC1-related signaling, including deteriorated energy status and suppressed mitochondrial oxidative function. In summary, we successfully established an in vitro skeletal muscle model of IOPD using patient-specific iPSCs. Disturbed mTORC1 signaling may contribute to the pathogenesis of skeletal muscle damage in IOPD, and may be a potential therapeutic target for Pompe disease.

Administration of perilla oil coated with Calshell increases glucagon-like peptide secretion.

Recently, we found that unsaturated long-chain fatty acids (such as alpha-linolenic acid) promote the secretion of glucagon-like peptide-1 (GLP-1) via G protein-coupled receptor GPR120, which is expressed predominantly in the colon. In order to ensure that the triglycerides or free fatty acids, such as alpha-linolenic acid, reach the distal intestinal tract effectively, we developed a Calshell technique. Following single treatment of Calshell perilla oil powder, the GLP-1 secretion level was significantly higher than following vehicle treatment, 120 min after treatment. Next, we examined the effects of long-term Calshell perilla oil powder treatment on GLP-1 secretion. Plasma GLP-1 level of Calshell perilla oil powder treatment was significantly higher than of vehicle treatment for 1, 14, 28 and 56 d. We thereby demonstrated for the first time the utility of Calshell oil powder treatment for effective and sustainable GLP-1 secretion. The Calshell technique is apparently useful as a drug delivery system, since Calshell unsaturated oil powder is protected from gastric acid, reaches enteroendocrine cells in the gastrointestinal tract, and then induces effective incretin secretion.