Physiological and ultrastructural features of human induced pluripotent and embryonic stem cell-derived skeletal myocytes in vitro

Abstract

Progress has recently been made toward the production of human skeletal muscle cells from induced pluripotent stem (iPS) cells. However, the functional and ultrastructural characterization, which is crucial for disease modeling and drug discovery, remains to be documented. We show, for the first time to our knowledge, that the electrophysiological properties of human iPS-derived skeletal myocytes are strictly similar to those of their embryonic stem (ES) cell counterparts, and both are typical of aneural mammalian skeletal muscle. In both cell types, intracellular calcium signaling that links membrane depolarization to contraction occurs in the absence of extracellular Ca 2+, a unique feature of skeletal muscle. Detailed analysis of the Ca 2+ signal revealed diverse kinetics of the rising phase, and hence various rates in the release of Ca 2+ from the sarcoplasmic reticulum. This was mirrored by ultrastructural evidence of Ca 2+ release units, which varied in location, shape, and size. Thus, the excitation-contraction coupling machinery of both iPS-and ES-derived skeletal myocytes was functional and specific, but did not reach full maturity in culture. This is in contrast with the myofibrillar network, which displayed the same organization as in adult skeletal muscle. Overall, the present study validates the human iPS-based skeletal myocyte model in comparison with the embryonic system, and provides the functional and ultrastructural basis for its application to human skeletal muscle diseases.human iPS-myocyte | human ES-myocyte | electrophysiology | EC coupling | ultrastructure T he generation of induced pluripotent stem (iPS) cells by genetic reprogramming of adult human somatic cells has opened great opportunities for basic research and regenerative medicine. Modeling human diseases with iPS cell technology offers a direct, noninvasive, and renewable experimental system for reproducing and studying pathological conditions. Within 5-6 y after the first report on human iPS cells (1), an amazing number of diseases affecting various systems (neurological, metabolic, cardiovascular, hematopoietic) have been modeled by reprogramming patient somatic cells (especially skin fibroblasts) into iPS cells followed by specific differentiation into cell types affected by the disease (2). Nevertheless, the efficiency of generating a robust population of cell progenitors from human iPS cells with a high differentiation potential in culture varies widely between different tissues. In particular, producing skeletal muscle cells from iPS cells turned out to be challenging, as reflected by the very limited number of reports and laboratories with successful results. This is in contrast to cardiac muscle, which has widely benefited from iPS cell technology, as various hereditary heart diseases have been modeled and explored (3, 4). We have previously shown that inducible expression of paired box (PAX) 3 or 7 transcription factors in both murine embryonic stem (ES) and iPS cells promotes the production of myogenic progenitors (5, 6)...

Making muscle: skeletal myogenesisin vivoandin vitro

“…While these studies are encouraging, ultrastructural analyses have shown that hPSC-derived myofibers have myofibrils that are less regularly organized than those found in vivo, featuring irregular and misaligned Z-disks. They also fail to form a mature T-tubule network, a hallmark of adult myofibers (Skoglund et al, 2014). Although triad-like structures could be identified, they were limited to the subsarcolemmal space, lacking the orthogonal alignment to myofibrils observed in vivo.…”

“…Remarkably, the formation of such long striated myofibers is rarely observed when primary myoblasts, myogenic lines (such as C2C12) or satellite cells are differentiated in vitro, as they tend to form poorly organized multinucleated myosacs. Electron microscopy analysis of mouse and human PSC-derived myofibers revealed that they exhibit highly organized sarcomeric units, reminiscent of the cytoarchitecture found in vivo (Hirsch et al, 1998;Skoglund et al, 2014). Myofibrils generated in these fibers can exhibit the appropriate organization of MyHC, troponin T, nebulin and titin, and undergo the stage-specific developmental transitions through α-actins (cardiac to skeletal) and MyHC (embryonic, fetal, and perinatal/fast) isoforms (Chal et al, 2015;Hirsch et al, 1998;Mizuno et al, 2009;Rohwedel et al, 1998b).…”

“…Mature striated myotubes have been generated in vitro from mESCs spontaneously differentiated from EBs (Hirsch et al, 1998;Rohwedel et al, 1998b), as well as from Pax7-reprogrammed hPSCs (Skoglund et al, 2014). Demestre et al (2015) compared these two methods in their ability to produce human actinin + myotubes and reported that myotubes generated by Pax7-mediated reprogramming were slower to mature.…”

“…In vivo, early embryonic myofibers exhibit transient, low voltageactivated calcium currents (T-type, ICaT), which are progressively replaced during fetal stages by slow, high voltage-activated currents (L-type, ICaL) (Beam et al, 1986;Cognard et al, 1986). Myofibers derived from Pax7-reprogrammed hPSCs exhibit excitable properties (Skoglund et al, 2014) but have immature ion handling characterized by slow calcium buffering and a small potassium conductance, which could explain the spontaneous fiber twitches observed in these cultures (Skoglund et al, 2014).…”

Formation and characterisation of neuromuscular junctions between hiPSC derived motoneurons and myotubes

“…Second, the generation of hiPSC-derived myoblasts by PAX7 over expression was done as previously described by Darabi and colleagues (Darabi et al, 2012;Skoglund et al, 2014). Details are described in supplemental methods.…”