Abstract
The rapid progress of research involving pluripotent stem cells in recent years provides an unprecedented opportunity to develop new and efficient cell replacement therapies for the treatment of degenerative diseases, such as muscular dystrophies. Nevertheless, the directed differentiation of pluripotent stem cells into a specific lineage is challenging, particularly the skeletal myogenic lineage which is poorly recapitulated during the in vitro differentiation of pluripotent stem cells. As demonstrated by our previous work, this limitation can be overcome by controlled expression of PAX7, which forces the commitment and expansion of human skeletal myogenic progenitors. Since variation in paraxial mesoderm formation among different human pluripotent stem cell lines/clones can affect the ability of PAX7 to induce the skeletal myogenic lineage, here we describe the optimization of our previously published protocol for deriving skeletal myogenic progenitors from these cells. This method takes advantage of the ability of the GSK3β inhibitor to induce paraxial mesoderm commitment in differentiating cells. In addition, upon flow cytometry purification, human PAX7-induced skeletal myogenic progenitors can be propagated and differentiated into skeletal myotubes. The following protocol will enable researchers to derive skeletal myogenic progenitors from pluripotent stem cells for disease modeling as wells as therapeutic purposes.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Arpke RW, Darabi R, Mader TL, Zhang Y, Toyama A, Lonetree CL, Nash N, Lowe DA, Perlingeiro RC, Kyba M (2013) A new immuno-, dystrophin-deficient model, the NSG-mdx(4Cv) mouse, provides evidence for functional improvement following allogeneic satellite cell transplantation. Stem Cells 31(8):1611–1620
Barberi T, Bradbury M, Dincer Z, Panagiotakos G, Socci ND, Studer L (2007) Derivation of engraftable skeletal myoblasts from human embryonic stem cells. Nat Med 13(5):642–648
Borchin B, Chen J, Barberi T (2013) Derivation and FACS-mediated purification of PAX3+/PAX7+ skeletal muscle precursors from human pluripotent stem cells. Stem Cell Rep 1(6):620–631
Chal J, Oginuma M, Al Tanoury Z, Gobert B, Sumara O, Hick A, Bousson F, Zidouni Y, Mursch C, Moncuquet P, Tassy O, Vincent S, Miyanari A, Bera A, Garnier JM, Guevara G, Hestin M, Kennedy L, Hayashi S, Drayton B, Cherrier T, Gayraud-Morel B, Gussoni E, Relaix F, Tajbakhsh S, Pourquie O (2015) Differentiation of pluripotent stem cells to muscle fiber to model Duchenne muscular dystrophy. Nat Biotechnol 33(9):962–969
Chang H, Yoshimoto M, Umeda K, Iwasa T, Mizuno Y, Fukada S-I, Yamamoto H, Motohashi N, Miyagoe-Suzuki Y, Takeda SI, Heike T, Nakahata T (2009) Generation of transplantable, functional satellite-like cells from mouse embryonic stem cells. FASEB J 23(6):1907–1919
Darabi R, Perlingeiro RC (2016) Derivation of skeletal myogenic precursors from human pluripotent stem cells using conditional expression of PAX7. In: Turksen K, Nagy A (eds) Induced Pluripotent Stem (iPS) Cells: Methods and Protocols, Springer, New York, p 423–439
Darabi R, Gehlbach K, Bachoo RM, Kamath S, Osawa M, Kamm KE, Kyba M, Perlingeiro RC (2008) Functional skeletal muscle regeneration from differentiating embryonic stem cells. Nat Med 14(2):134–143
Darabi R, Arpke RW, Irion S, Dimos JT, Grskovic M, Kyba M, Perlingeiro RC (2012) Human ES- and iPS-derived myogenic progenitors restore DYSTROPHIN and improve contractility upon transplantation in dystrophic mice. Cell Stem Cell 10(5):610–619
Garriock RJ, Chalamalasetty RB, Kennedy MW, Canizales LC, Lewandoski M, Yamaguchi TP (2015) Lineage tracing of neuromesodermal progenitors reveals novel Wnt-dependent roles in trunk progenitor cell maintenance and differentiation. Development 142(9):1628–1638
Maroto M, Reshef R, Munsterberg AE, Koester S, Goulding M, Lassar AB (1997) Ectopic Pax-3 activates MyoD and Myf-5 expression in embryonic mesoderm and neural tissue. Cell 89(1):139–148
Mendjan S, Mascetti VL, Ortmann D, Ortiz M, Karjosukarso DW, Ng Y, Moreau T, Pedersen RA (2014) NANOG and CDX2 pattern distinct subtypes of human mesoderm during exit from pluripotency. Cell Stem Cell 15(3):310–325
Rinaldi F, Perlingeiro RC (2014) Stem cells for skeletal muscle regeneration: therapeutic potential and roadblocks. Transl Res 163(4):409–417
Shelton M, Metz J, Liu J, Carpenedo RL, Demers SP, Stanford WL, Skerjanc IS (2014) Derivation and expansion of PAX7-positive muscle progenitors from human and mouse embryonic stem cells. Stem Cell Rep 3(3):516–529
Tam PP, Loebel DA (2007) Gene function in mouse embryogenesis: get set for gastrulation. Nat Rev Genet 8(5):368–381
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
Copyright information
© 2016 Springer International Publishing Switzerland
About this chapter
Cite this chapter
Kim, J., Magli, A., Perlingeiro, R.C.R. (2016). Efficient Generation of Skeletal Myogenic Progenitors from Human Pluripotent Stem Cells. In: Working with Stem Cells. Springer, Cham. https://doi.org/10.1007/978-3-319-30582-0_16
Download citation
DOI: https://doi.org/10.1007/978-3-319-30582-0_16
Published:
Publisher Name: Springer, Cham
Print ISBN: 978-3-319-30580-6
Online ISBN: 978-3-319-30582-0
eBook Packages: Biomedical and Life SciencesBiomedical and Life Sciences (R0)