Self-Organizing 3D Human Trunk Neuromuscular Organoids

Martins, Jorge-Miguel Faustino; Fischer, Cornelius; Urzi, Alessia; Vidal, Ramón; Kunz, Séverine; Ruffault, Pierre-Louis; Kabuss, Loreen; Hube, Iris; Gazzerro, Elisabeta; Birchmeier, Carmen; Spuler, Simone; Sauer, Sascha; Gouti, Mina

doi:10.1016/j.stem.2019.12.007

Cited by 229 publications

(181 citation statements)

References 73 publications

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“…Albeit based on the RNA seq data, the fetal acetylcholine receptor subunit isoform was more abundant than the adult isoform at D30 postdifferentiation. A recent article reported the production of human trunk neuromuscular organoids in which maturity was reached at day 50 postdifferentiation [42]. Interestingly, using a different approach, we reached the same conclusion, i.e., that the simultaneous differentiation of muscle and peripheral neurons is necessary for the production of mature and functional muscle fibers, an important prerequisite for disease modeling.…”

Section: Discussionsupporting

confidence: 73%

“…Furthermore, as genome-wide approaches often prove difficult regarding the interpretation of variants of unknown significance (VUS), this protocol provides a novel tool for the evaluation and deciphering of pathways associated with diseases causing mutations and associated signatures. Thus, compared to the occasional twitching reported for control cells [7,32], or the absence of twitching in patient cells [32], our straightforward procedure fills the gap between currently available 2D protocols, in which maturity and functionality are reduced, and the advances provided by neuromuscular organoids [42]. Compared to organoids, contractions were visible as early as 19-21 days postdifferentiation in our conditions.…”

Section: Discussionmentioning

confidence: 70%

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Multilineage Differentiation for Formation of Innervated Skeletal Muscle Fibers from Healthy and Diseased Human Pluripotent Stem Cells

Mazaleyrat

Badja

Broucqsault

et al. 2020

Cells

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Induced pluripotent stem cells (iPSCs) obtained by reprogramming primary somatic cells have revolutionized the fields of cell biology and disease modeling. However, the number protocols for generating mature muscle fibers with sarcolemmal organization using iPSCs remain limited, and partly mimic the complexity of mature skeletal muscle. Methods: We used a novel combination of small molecules added in a precise sequence for the simultaneous codifferentiation of human iPSCs into skeletal muscle cells and motor neurons. Results: We show that the presence of both cell types reduces the production time for millimeter-long multinucleated muscle fibers with sarcolemmal organization. Muscle fiber contractions are visible in 19–21 days, and can be maintained over long period thanks to the production of innervated multinucleated mature skeletal muscle fibers with autonomous cell regeneration of PAX7-positive cells and extracellular matrix synthesis. The sequential addition of specific molecules recapitulates key steps of human peripheral neurogenesis and myogenesis. Furthermore, this organoid-like culture can be used for functional evaluation and drug screening. Conclusion: Our protocol, which is applicable to hiPSCs from healthy individuals, was validated in Duchenne Muscular Dystrophy, Myotonic Dystrophy, Facio-Scapulo-Humeral Dystrophy and type 2A Limb-Girdle Muscular Dystrophy, opening new paths for the exploration of muscle differentiation, disease modeling and drug discovery.

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Section: Discussionsupporting

confidence: 73%

Section: Discussionmentioning

confidence: 70%

Multilineage Differentiation for Formation of Innervated Skeletal Muscle Fibers from Healthy and Diseased Human Pluripotent Stem Cells

Mazaleyrat

Badja

Broucqsault

et al. 2020

Cells

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“…Our data further supported that organoid culture represented fetal stages as we could detect NFIX upregulation and verify presence of myofibers expressing fetal MyHC isoform as well as NCAM, M-Cad or MCAM in the proximity of PAX7 progenitors (Figure 3G to J). Figures 2F, 3A), while in current 2D or 3D protocols there is a constant decline of the myogenic progenitor reservoir (Chal et al, 2015, Faustino-Martins et al, 2020 (Figure 5 -figure supplement 5). Upregulation of the committed myogenic marker MYOD1 in current protocols could explain the constant progenitor decline over time, as well as their more undifferentiated state.…”

Section: Discussionmentioning

confidence: 83%

“…Subsequently surface marker combinations could be used to isolate of myogenic progenitors with in vivo repopulation potential (Magli et al, 2017, Hicks et al, 2018, Al Tanoury et al, 2020. A few three-dimensional (3D) differentiation approaches provide cohorts of terminally differentiated myofibers and focus their investigation on potential interactions with the vasculature and nervous system without evaluating the developmental identity or sustainability of myogenic progenitors (Faustino Martins et al, 2020, Maffioletti et al, 2018, Rao et al, 2018. Single cell technologies increasingly provide databases for deciphering myogenic trajectories and expression profiles of myogenic stem and progenitor cells (Xi et al, 2020) and PSC differentiation protocols can be evaluated in their potency to mimic human development.…”

Section: Introductionmentioning

confidence: 99%

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

Mavrommatis

Jeong

Gomez-Giro

et al. 2020

Preprint

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In vitro culture systems which structurally recapitulate human myogenesis and promote PAX7+ myogenic progenitor maturation have not been established. Here we report human skeletal muscle organoids differentiated from induced pluripotent stem cell lines that contain paraxial mesoderm and neuromesodermal progenitors and develop into organized structures, reassembling neural plate border and dermomyotome formation. Culture conditions causes neural lineage arrest and promotes fetal hypaxial myogenesis towards limb axial anatomical identity and generates sustainable uncommitted PAX7 myogenic progenitors, as well as fibroadipogenic (PDGFRa+) progenitor populations equivalent to those from second trimester of human gestation. Single cell comparison to human fetal and adult myogenic progenitors, reveals distinct molecular signatures for myogenic progenitors in activated (CD44+, CD98+, MYOD1+, MYF5+) and in dormant (PAX7+, FBN1+, SPRY1+, CHODL1+) states, as well as indicated developmental trajectories associated to myogenic commitment and differentiation or self-renewal. Our approach, further validated with Duchenne and CRISPR/Cas9 genome-edited Limb-girdle muscular dystrophy (LGMD2A) patient iPSC lines, provides a robust in vitro developmental system for investigating muscle tissue morphogenesis and homeostasis.

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“…The development of multi-lineage organoids opens new avenues to obtain personalized micro-physiological models for modeling developmental organogenesis in vitro . Few examples include the fusion of neural spheroids or cortical organoids ( Bagley et al., 2017 ; Birey et al., 2017 ; Xiang et al., 2017 , 2019 ), generating hybrid neuromuscular organoids ( Faustino Martins et al., 2020 ), fusing anterior and foregut endoderm for hepato-biliary and pancreatic organoid structures ( Koike et al., 2019 ), and generating intestinal organoids with a functional enteric nervous system ( Workman et al., 2017 ). Multi-lineage organoids generated from co-emerging cardiac and gut lineage specification from iPSCs can be useful to model heart complexity better than conventional cardiac organoids.…”

Section: Mimicking Development To Generate Multi-lineage Organoidsmentioning

confidence: 99%

Translating Embryogenesis to Generate Organoids: Novel Approaches to Personalized Medicine

Sahu

Sharan

2020

iScience

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Summary The astounding capacity of pluripotent stem cells (PSCs) to differentiate and self-organize has revolutionized the development of 3D cell culture models. The major advantage is its ability to mimic in vivo microenvironments and cellular interactions when compared with the classical 2D cell culture models. Recent innovations in generating embryo-like structures (including blastoids and gastruloids) from PSCs have advanced the experimental accessibility to understand embryogenesis with immense potential to model human development. Taking cues on how embryonic development leads to organogenesis, PSCs can also be directly differentiated to form mini-organs or organoids of a particular lineage. Organoids have opened new avenues to augment our understanding of stem cell and regenerative biology, tissue homeostasis, and disease mechanisms. In this review, we provide insights from developmental biology with a comprehensive resource of signaling pathways that in a coordinated manner form embryo-like structures and organoids. Moreover, the advent of assembloids and multilineage organoids from PSCs opens a new dimension to study paracrine function and multi-tissue interactions in vitro . Although this led to an avalanche of enthusiasm to utilize organoids for organ transplantation studies, we examine the current limitations and provide perspectives to improve reproducibility, scalability, functional complexity, and cell-type characterization. Taken together, these 3D in vitro organ-specific and patient-specific models hold great promise for drug discovery, clinical management, and personalized medicine.

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Self-Organizing 3D Human Trunk Neuromuscular Organoids

Abstract: Highlights d hPSC-derived neuromesodermal progenitors generate functional NMOs in 3D d Functional NMJs are generated in NMOs supported by terminal Schwann cells d NMOs contract and develop central pattern generator-like circuits d NMOs can be used to model key aspects of myasthenia gravis

Cited by 229 publications

References 73 publications

Multilineage Differentiation for Formation of Innervated Skeletal Muscle Fibers from Healthy and Diseased Human Pluripotent Stem Cells

Multilineage Differentiation for Formation of Innervated Skeletal Muscle Fibers from Healthy and Diseased Human Pluripotent Stem Cells

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

Translating Embryogenesis to Generate Organoids: Novel Approaches to Personalized Medicine

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