Generation of Skeletal Muscle Organoids from Human Pluripotent Stem Cells.

Various protocols have been proven effective in the directed differentiation of mouse and human pluripotent stem cells into skeletal muscles and used to study myogenesis. Current 2D myogenic differentiation protocols can mimic muscle development and its alteration under pathological conditions such as muscular dystrophies. 3D skeletal muscle differentiation approaches can, in addition, model the interaction between the various cell types within the developing organoid. Our protocol ensures the differentiation of human embryonic/induced pluripotent stem cells (hESC/hiPSC) into skeletal muscle organoids (SMO) via cells with paraxial mesoderm and neuromesodermal progenitors' identity and further production of organized structures of the neural plate margin and the dermomyotome. Continuous culturing omits neural lineage differentiation and promotes fetal myogenesis, including the maturation of fibroadipogenic progenitors and PAX7-positive myogenic progenitors. The PAX7 progenitors resemble the late fetal stages of human development and, based on single-cell transcriptomic profiling, cluster close to adult satellite cells of primary muscles. To overcome the limited availability of muscle biopsies from patients with muscular dystrophy during disease progression, we propose to use the SMO system, which delivers a stable population of skeletal muscle progenitors from patient-specific iPSCs to investigate human myogenesis in healthy and diseased conditions. Key features • Development of skeletal muscle organoid differentiation from human pluripotent stem cells, which recapitulates myogenesis. • Analysis of early embryonic and fetal myogenesis. • Provision of skeletal muscle progenitors for in vitro and in vivo analysis for up to 14 weeks of organoid culture. • In vitro myogenesis from patient-specific iPSCs allows to overcome the bottleneck of muscle biopsies of patients with pathological conditions.

Human skeletal muscle organoids model fetal myogenesis and sustain uncommitted PAX7 myogenic progenitors.

In vitro culture systems that structurally model human myogenesis and promote PAX7+ myogenic progenitor maturation have not been established. Here we report that human skeletal muscle organoids can be differentiated from induced pluripotent stem cell lines to contain paraxial mesoderm and neuromesodermal progenitors and develop into organized structures reassembling neural plate border and dermomyotome. Culture conditions instigate neural lineage arrest and promote fetal hypaxial myogenesis toward limb axial anatomical identity, with generation of sustainable uncommitted PAX7 myogenic progenitors and fibroadipogenic (PDGFRa+) progenitor populations equivalent to those from the second trimester of human gestation. Single-cell comparison to human fetal and adult myogenic progenitor /satellite cells reveals distinct molecular signatures for non-dividing myogenic progenitors in activated (CD44High/CD98+/MYOD1+) and dormant (PAX7High/FBN1High/SPRY1High) states. Our approach provides a robust 3D in vitro developmental system for investigating muscle tissue morphogenesis and homeostasis.

Humans contains around 650 skeletal muscles which allow the body to move around and maintain its posture. Skeletal muscles are made up of individual cells that bundle together into highly organized structures. If this group of muscles fail to develop correctly in the embryo and/or fetus, this can lead to muscular disorders that can make it painful and difficult to move. One way to better understand how skeletal muscles are formed, and how this process can go wrong, is to grow them in the laboratory. This can be achieved using induced pluripotent stem cells (iPSCs), human adult cells that have been 'reprogrammed' to behave like cells in the embryo that can develop in to almost any cell in the body. The iPSCs can then be converted into specific cell types in the laboratory, including the cells that make up skeletal muscle. Here, Mavrommatis et al. created a protocol for developing iPSCs into three-dimensional organoids which resemble how cells of the skeletal muscle look and arrange themselves in the fetus. To form the skeletal muscle organoid, Mavrommatis et al. treated iPSCs that were growing in a three-dimensional environment with various factors that are found early on in development. This caused the iPSCs to organize themselves in to embryonic and fetal structures that will eventually give rise to the parts of the body that contain skeletal muscle, such as the limbs. Within the organoid were cells that produced Pax7, a protein commonly found in myogenic progenitors that specifically mature into skeletal muscle cells in the fetus. Pax 7 is also present in 'satellite cells' that help to regrow damaged skeletal muscle in adults. Indeed, Mavrommatis et al. found that the myogenic progenitors produced by the organoid were able to regenerate muscle when transplanted in to adult mice. These findings suggest that this organoid protocol can generate cells that will give rise to skeletal muscle. In the future, these lab-grown progenitors could potentially be created from cells isolated from patients and used to repair muscle injuries. The organoid model could also provide new insights in to how skeletal muscles develop in the fetus, and how genetic mutations linked with muscular disorders disrupt this process.

CRISPR/Cas9 Genome Editing in LGMD2A/R1 Patient-Derived Induced Pluripotent Stem and Skeletal Muscle Progenitor Cells.

Large numbers of Calpain 3 (CAPN3) mutations cause recessive forms of limb-girdle muscular dystrophy (LGMD2A/LGMDR1) with selective atrophy of the proximal limb muscles. We have generated induced pluripotent stem cells (iPSC) from a patient with two mutations in exon 3 and exon 4 at the calpain 3 locus (W130C, 550delA). Two different strategies to rescue these mutations are devised: (i) on the level of LGMD2A-iPSC, we combined CRISPR/Cas9 genome targeting with a FACS and Tet transactivator-based biallelic selection strategy, which resulted in a new functional chimeric exon 3-4 without the two CAPN3 mutations. (ii) On the level of LGMD2A-iPSC-derived CD82+/Pax7+ myogenic progenitor cells, we demonstrate CRISPR/Cas9 mediated rescue of the highly prevalent exon 4 CAPN3 mutation. The first strategy specifically provides isogenic LGMD2A corrected iPSC for disease modelling, and the second strategy can be further elaborated for potential translational approaches.

Detection of Math6-Expressing Cell Types in Murine Placenta.

The transcription factor Math6, mouse atonal homolog 6, belongs to the family of highly conserved basic helix-loop-helix transcription factors. It plays an important role in embryonic development and shows a wide expression pattern in murine tissues. The placenta, as a life-sustaining transient organ for the fetus, also depends on the expression of Math6. The adverse effects of deleting Math6 in mice, leading to deficient placental development and pregnancy loss, have already been demonstrated by us. Until now, detailed investigations regarding the specific mechanisms underlying the improper placental development in these murine mutants have failed, as the Math6 expression could not be confined to a specific cell type due to the lack of a highly specific Math6 antibody. To circumvent this problem, we used transgenic mice, where Math6 is marked with a Flag sequence that functions as a specific epitope. Tissues from these transgenic mice were used to establish immunohistochemical staining and fluorescence-activated cell sorting (FACS). The establishment of these methods yielded initial findings pertaining to the identification of Math6-expressing cell types and their localization. Our results reveal that Math6 shows a wide expression pattern in both maternal and fetal components of the murine placenta. It shows expression in various cell types, but predominantly in trophoblast giant cells, endothelial cells and macrophages. The largest subpopulation that we detected in the group of Math6-positive cells were identified as DBA+ uterine natural killer cells. These findings reveal information and a chance for further investigation on the involvement of Math6 in placental development and the molecular pathomechanisms of spontaneous abortion.

Athymic mice reveal a requirement for T-cell-microglia interactions in establishing a microenvironment supportive of Nf1 low-grade glioma growth.

Pediatric low-grade gliomas (LGGs) frequently do not engraft in immunocompromised mice, limiting their use as an experimental platform. In contrast, murine Neurofibromatosis-1 (Nf1) optic LGG stem cells (o-GSCs) form glioma-like lesions in wild-type, but not athymic, mice following transplantation. Here, we show that the inability of athymic mice to support o-GSC engraftment results from impaired microglia/macrophage function, including reduced expression of Ccr2 and Ccl5, both of which are required for o-GSC engraftment and Nf1 optic glioma growth. Impaired Ccr2 and Ccl5 expression in athymic microglia/macrophages was restored by T-cell exposure, establishing T-cell-microglia/macrophage interactions as critical stromal determinants that support NF1 LGG growth.