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

peer reviewedIn 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

Human skeletal muscle organoids recapitulate myogenesis, provide axial anatomical identity and generate distinct dystrophic phenotypes

In dieser Studie beschreiben wir, dass durch Differenzierung humaner pluripotenter Stammzellen in einer 3D-Umgebung verschiedene Stadien der menschlichen Myogenese als "Skelettmuskelorganoide" strukturell reproduziert werden können. Zu diesen Stadien gehören die Entwicklung des paraxialen Mesoderms und des Dermomyotoms sowie die Delaminierung und die Migration des Dermomyotoms. Wir konnten funktionell ausgereiften Skelettmuskel differenzieren, der Pax7$^{+}$ Zellen unter seiner Basallamina für mindestens 14 Wochen konserviert. Durch Applikation von CRISPR/Cas9 Genom Editing konnten wir zwei Gendefekte in benachbarten Exons des Calpain 3 Gens in Gliedergürtelmuskeldystrophie Patienten- spezifischen induzierten pluripotenten Stammzelle korrigieren. Hierdurch konnte die eingeschränkte Membranintegrität wiederhergestellt werden. Differenzierung von Duchenne Muskeldystrophie Patienten-spezifischen iPS Zellen in unserem Kulturmodell weisen eine Entwicklungsinhibition in Vorläuferstadien auf

Generation of a human iPSC line (HIMRi001-A) from a patient with filaminopathy

Here we introduce the human induced pluripotent stem cell (hiPSC) line HIMRi001-A generated from cultured dermal fibroblasts of a 60-year-old male patient with a myofibrillar myopathy, carrying a heterozygous c.4984C > T [p.Q1662X] mutation in the $\textit {filamin C}$ ($\it FLNC$)-gene, via lentiviral expression of OCT4, SOX2, KLF4 and c-MYC. HIMRi001-A displays typical embryonic stem cell-like morphology, carries the c.4984C > T $\it FLNC$ gene mutation, expressed several pluripotent stem cell makers, retained normal karyotype (46, XY) and holds the potential to differentiate in all three germ layers. We postulate that HIMRi001-A can be used for the elucidation of $\it FLNC$-associated pathomechanisms and for developing new therapeutic options

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

Generation of two human iPSC lines (HIMRi002-A and HIMRi003-A) derived from Caveolinopathy patients with rippling muscle disease

Here we introduce the human induced pluripotent stem cell lines (hiPSCs), HIMRi002-A and HIMRi003-A, generated from cultured dermal fibroblasts of 61-year-old (HIMRi002-A) and 38-year-old (HIMRi003-A) female patients, carrying a known heterozygous pathogenic variant ($\it p.A46T$) in the $\textit {Caveolin 3}$ ($\it CAV3$) gene, via lentiviral expression of OCT4, SOX2, KLF4 and c-MYC. HIMRi002-A and HIMRi003-A display typical embryonic stem cell-like morphology, carry the $\it p.A46T$ $\it CAV3$ gene mutation, express several pluripotent stem cell markers, retain normal karyotype (46, XX) and can differentiate in all three germ layers. We postulate that the HIMRi002-A and HIMRi003-A iPSC lines can be used for the characterization of $\it CAV3$-associated pathomechanisms and for developing new therapeutic options