Efficient derivation and inducible differentiation of expandable skeletal myogenic cells from human ES and patient-specific iPS cells

Abstract

Skeletal muscle is the most abundant human tissue; therefore, an unlimited availability of myogenic cells has applications in regenerative medicine and drug development. Here we detail a protocol to derive myogenic cells from human embryonic stem (ES) and induced pluripotent stem (iPS) cells, and we also provide evidence for its extension to human iPS cells cultured without feeder cells. The procedure, which does not require the generation of embryoid bodies or prospective cell isolation, entails four stages with different culture densities, media and surface coating. Pluripotent stem cells are disaggregated to single cells and then differentiated into expandable cells resembling human mesoangioblasts. Subsequently, transient Myod1 induction efficiently drives myogenic differentiation into multinucleated myotubes. Cells derived from patients with muscular dystrophy and differentiated using this protocol have been genetically corrected, and they were proven to have therapeutic potential in dystrophic mice. Thus, this platform has been demonstrated to be amenable to gene and cell therapy, and it could be extended to muscle tissue engineering and disease modeling.

Laminin 521 maintains differentiation potential of mouse and human satellite cell-derived myoblasts during long-term culture expansion

“…Studies are currently underway to analyze the engraftment potential of mouse myoblasts expanded on varying substrates including laminin 521. The differentiation of skeletal muscle differentiation from iPSCs is currently not well defined even though a number of reports have been published using protocols with varying techniques and ECM substrates [47–50]. Since laminin α2, laminin α4, and laminin α5 are expressed during muscle development, it will be important to test these substrates in iPSC studies to determine the optimal ECM induction substrate [10, 11].…”

Krüppel-like factors in mammalian stem cells and development

“…Conversely, overexpression of Klf2 or Klf4 is sufficient for induction of naive pluripotency (Blakeley et al, 2015). Despite these differences between mouse and human ESCs, the generation of human iPSCs was accomplished using the same factors as for mouse iPSCs: Oct4, Sox2, c-Myc and Klf4 (Kim et al, 2008;Maffioletti et al, 2015;Moad et al, 2013;Park et al, 2008;Takahashi et al, 2007).…”

Differentiation and sarcomere formation in skeletal myocytes directly prepared from human induced pluripotent stem cells using a sphere-based culture

“…While many protocols require cell sorting and/or rely on exogenous expression of myogenic genes such as PAX3, PAX7, and MYOD (Abujarour et al, 2014; Darabi et al, 2012; Maffioletti et al, 2015; Skoglund et al, 2014; Tanaka et al, 2013), more recent advances have been made with the application of small molecules and growth factors to directly promote myogenic differentiation from human iPSCs (Barberi et al, 2007; Borchin et al, 2013; Caron et al, 2016; Chal et al, 2016; Chal et al, 2015; Choi et al, 2016; Hosoyama et al, 2014; Hwang et al, 2013; Shelton et al, 2014; Xu et al, 2013). Our group recently reported a unique method for the derivation of myogenic progenitors from human pluripotent cells using free-floating spherical culture (Hosoyama et al, 2014).…”