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3D human induced pluripotent stem cell–derived bioengineered skeletal muscles for tissue, disease and therapy modeling

Nature Protocols volume 18, pages 1337–1376 (2023)Cite this article

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Abstract

Skeletal muscle is a complex tissue composed of multinucleated myofibers responsible for force generation that are supported by multiple cell types. Many severe and lethal disorders affect skeletal muscle; therefore, engineering models to reproduce such cellular complexity and function are instrumental for investigating muscle pathophysiology and developing therapies. Here, we detail the modular 3D bioengineering of multilineage skeletal muscles from human induced pluripotent stem cells, which are first differentiated into myogenic, neural and vascular progenitor cells and then combined within 3D hydrogels under tension to generate an aligned myofiber scaffold containing vascular networks and motor neurons. 3D bioengineered muscles recapitulate morphological and functional features of human skeletal muscle, including establishment of a pool of cells expressing muscle stem cell markers. Importantly, bioengineered muscles provide a high-fidelity platform to study muscle pathology, such as emergence of dysmorphic nuclei in muscular dystrophies caused by mutant lamins. The protocol is easy to follow for operators with cell culture experience and takes between 9 and 30 d, depending on the number of cell lineages in the construct. We also provide examples of applications of this advanced platform for testing gene and cell therapies in vitro, as well as for in vivo studies, providing proof of principle of its potential as a tool to develop next-generation neuromuscular or musculoskeletal therapies.

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Fig. 1: Workflow to generate and analyse 3D skeletal muscle constructs using hiPSC-derived progenitors.
Fig. 2: Pilot testing and quality control for biomaterials and cells.
Fig. 3: Characterization of hiPSC-derived 3D bioengineered muscles.
Fig. 4: Modeling muscular dystrophies caused by abnormal nuclear envelope by using hiPSC-derived 3D muscles: stepwise 3D nuclear reconstruction of hiPSC-derived healthy control and LMNA-mutant skeletal myotubes in monolayer versus 3D (single-lineage) artificial muscles.
Fig. 5: Examples of in vitro and in vivo applications of hiPSC-derived 3D artificial muscles: testing gene therapy vectors, assessing cell migration capacity and studying biocompatibility.

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Data availability

Key raw data linked to the original publications30,31,54 reported in this protocol have been deposited in Mendeley Data and are available at https://doi.org/10.17632/d826fxhr3b.1. Raw data relative to Figs. 3c and 5b,c are available at https://doi.org/10.17632/hzmx5nkyff.1.

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Acknowledgements

We thank all co-authors of the original articles describing this technology, the EU FP7 projects Biodesign (262948) and Plurimes (602423) and G. Cossu, H. Redl and T. Eschenaghen for their initial support and contribution. We are grateful to CureCMD, Cellular Dynamics International, Inc., M. Oshimura and T. VandenDriessche for cell lines used in our original papers; to J. Ng for AAV9 vector particles; to the DSHB for providing MF20, F5D and titin antibodies; and to all laboratory members for helpful discussions. This work was supported by the European Research Council (759108–HISTOID); MDUK (19GRO-PS48-0188 and 17GRO-PS48-0093-1); the BBSRC London Interdisciplinary Biosciences Consortium (LIDo; BB/M009513/1 to F.S.T., P.S.Z., L.P. and N.K.); AFM-Telethon (21687 and 23782); Duchenne Parent Project NL (19.005); and the Francis Crick Institute, which receives its core funding from Cancer Research UK, the UK Medical Research Council (MRC) and the Wellcome Trust (CC0102). F.S.T. also acknowledges funding by the NIHR (CL-2018-18-008; the views expressed are those of the authors and not necessarily those of the National Health Service (NHS), the NIHR or the Department of Health). E.N. and A.S.B. acknowledge support from the Company of Biologists (DMMTF1905206) and Wellcome Trust (210987/Z/18/Z), respectively. The Zammit laboratory acknowledges the support of the MRC (MR/P023215/1 and MR/S002472/1), MDUK (19GRO-PG12-0493) and the FSHD Society. Work with human cells was performed under approval of the NHS Health Research Authority Research Ethics Committee (reference no. 13/LO/1826) and Integrated Research Application System (IRAS) project (ID No. 141100).

Author information

Author notes

  1. Sara M. Maffioletti

    Present address: San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), Milan, Italy

  2. These authors contributed equally: Luca Pinton, Moustafa Khedr.

Authors and Affiliations

  1. Department of Cell and Developmental Biology, University College London, London, UK

    Luca Pinton, Moustafa Khedr, Valentina M. Lionello, Shilpita Sarcar, Sara M. Maffioletti, Sumitava Dastidar, Elisa Negroni, SungWoo Choi, Noreen Khokhar & Francesco Saverio Tedesco

  2. The Francis Crick Institute, London, UK

    Luca Pinton, Moustafa Khedr, Valentina M. Lionello, Sumitava Dastidar, SungWoo Choi, Noreen Khokhar, Andreia Sofia Bernardo & Francesco Saverio Tedesco

  3. Randall Centre for Cell and Molecular Biophysics, King’s College London, London, UK

    Luca Pinton, Noreen Khokhar & Peter S. Zammit

  4. Center for Research in Myology UMRS974, Sorbonne Université, INSERM, Myology Institute AIM, Paris, France

    Elisa Negroni & Anne Bigot

  5. UCL Division of Surgery and Interventional Science, Royal Free Hospital, London, UK

    John R. Counsell

  6. Dubowitz Neuromuscular Centre, UCL Great Ormond Street Institute of Child Health and Great Ormond Street Hospital for Children, London, UK

    John R. Counsell & Francesco Saverio Tedesco

  7. National Heart and Lung Institute, Imperial College London, London, UK

    Andreia Sofia Bernardo

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Contributions

L.P., M.K. and F.S.T. wrote the paper, assembled the figures and performed the protocolization and optimization of all the steps listed. V.M.L. and S.S. contributed equally to this work. L.P. performed disease modeling experiments and analysis. M.K. performed calcium experiments and advanced imaging. M.K. and S.S. performed AAV experiments with support from J.R.C. V.M.L. performed contractility and calcium experiments with support from A.S.B. S.D. and S.W.C. performed (Fig. 5a,c) experiments, respectively. S.M.M. and S.S. developed the 3D skeletal muscle platform with F.S.T. and discussed results. N.K. derived neural progenitor cells. E.N. performed work on immortalized myoblasts provided by A.B. P.S.Z. co-supervised L.P. with F.S.T. during his PhD research, contributed to his funding and reviewed manuscript drafts. F.S.T. provided funding, coordinated the work, discussed and analyzed results, wrote and reviewed manuscript drafts with all co-authors and finalized the paper.

Corresponding author

Correspondence to Francesco Saverio Tedesco.

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Competing interests

F.S.T. has received speaker and consultancy honoraria from Takeda, Sanofi Genzyme and Aleph Farms (via UCL Consultants). All other authors declare no competing interests.

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Related links

Key references using this protocol

Maffioletti, S. M. et al. Cell Rep. 23, 899–908 (2018): https://doi.org/10.1016/j.celrep.2018.03.091

Steele-Stallard, H. B. et al. Front. Physiol. 9, 1332 (2018): https://doi.org/10.3389/fphys.2018.01332

Choi, S. et al. EMBO Mol. Med. 14, e14526 (2022): https://doi.org/10.15252/emmm.202114526

Supplementary information

Supplementary Information

Supplementary Table 1 and Protocol 1

Supplementary Video 1

Real-time video of DMD hiPSC-derived 3D skeletal muscle construct contractions in response to 10-V electrical stimulation at 1 Hz

Supplementary Video 2

Calcium transients in DMD hiPSC-derived 3D artificial muscles incubated with the fluorescence Ca2+ indicator Fluo-4AM upon electrical stimulation. During the first 10 s, no electrical stimuli were applied to the muscle. At second 10, the 3D muscle was stimulated with 10 V at 0.33 Hz, and at second 20, the 3D muscle was stimulated with 20 V at 0.33 Hz

Supplementary Video 3

Maximum-intensity projection time-lapse video of 5-chloromethylfluorescein diacetate (CMFDA)-stained HIDEMs (green) deposited on hiPSC-derived 3D muscles (red). 100-µm stacks of muscles were imaged every 12 min for 4 h by using a spinning disk microscope. Scale bar, 100 µm

Supplementary Video 4

3D reconstruction of a 3D artificial muscle imaged with a light-sheet microscope immunostained as detailed in the advanced imaging section of Fig. 2

Supplementary Video 5

3D reconstruction nuclear morphology (lamin A/C in red) and myotubes (eMyHC in green) of a healthy control hiPSC-derived 3D muscle construct shown in Fig. 3. The 3D reconstruction was done by using Imaris software and confocal z-stacks of immunostained artificial muscles. Video reproduced from ref. 29 under Creative Commons license CC BY 4.0

Supplementary Video 6

3D reconstruction nuclear morphology (lamin A/C in red) and myotubes (eMyHC in green) of an LMNA-mutant (R249W) hiPSC-derived 3D muscle construct shown in Fig. 3. The 3D reconstruction was done by using Imaris software and confocal z-stacks of immunostained artificial muscles. Video reproduced from ref. 29 under Creative Commons license CC BY 4.0

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Pinton, L., Khedr, M., Lionello, V.M. et al. 3D human induced pluripotent stem cell–derived bioengineered skeletal muscles for tissue, disease and therapy modeling. Nat Protoc 18, 1337–1376 (2023). https://doi.org/10.1038/s41596-022-00790-8

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