hiPSC-Derived Schwann Cells Influence Myogenic Differentiation in Neuromuscular Cocultures

Abstract

Motoneurons, skeletal muscle fibers, and Schwann cells form synapses, termed neuromuscular junctions (NMJs). These control voluntary body movement and are affected in numerous neuromuscular diseases. Therefore, a variety of NMJ in vitro models have been explored to enable mechanistic and pharmacological studies. So far, selective integration of Schwann cells in these models has been hampered, due to technical limitations. Here we present robust protocols for derivation of Schwann cells from human induced pluripotent stem cells (hiPSC) and their coculture with hiPSC-derived motoneurons and C2C12 muscle cells. Upon differentiation with tuned BMP signaling, Schwann cells expressed marker proteins, S100b, Gap43, vimentin, and myelin protein zero. Furthermore, they displayed typical spindle-shaped morphologies with long processes, which often aligned with motoneuron axons. Inclusion of Schwann cells in coculture experiments with hiPSC-derived motoneurons and C2C12 myoblasts enhanced myotube growth and affected size and number of acetylcholine receptor plaques on myotubes. Altogether, these data argue for the availability of a consistent differentiation protocol for Schwann cells and their amenability for functional integration into neuromuscular in vitro models, fostering future studies of neuromuscular mechanisms and disease.

Development and In Vitro Differentiation of Schwann Cells

“…In general, however, the neurosphere induction technique resulted in mixed neural precursor cultures and, thus, necessitated purification steps before ensuing differentiation. In comparison, the monolayer TGFβ block/Wnt activation approach can yield sufficient quantities of neural crest cells, without the need for purification methods [ 118 , 119 , 120 , 121 ].…”

“…With this method, consistent results of robust neural crest induction across different hPSC lines were achieved. Moreover, integration of this approach into a Schwann cell differentiation protocol led to an increased yield and robustness of neural crest induction [ 118 ]. In the future, similar signaling-tuning approaches may further aid in neural crest in vitro differentiation and in developing more effective and robust Schwann cell differentiation protocols.…”

“…Additionally, in some hiPSC/hESC protocols, a combination of CNTF, db-cAMP, and Nrg1 with bFGF [ 114 , 117 ] or without [ 116 ] was used, while one protocol applied only db-cAMP, ascorbic acid, and FBS [ 119 ]. Unfortunately, the presence of FBS or serum derivatives is still necessary in most of these protocols, at least for some phases of differentiation, and completely serum-free cultures lead to low cell survival [ 118 , 121 ]. Although serum does not seem to be required for cell propagation in primary human Schwann cell cultures, it is regularly used to promote their survival [ 19 ].…”

“…Nevertheless, CNTF was shown to induce Krox20 in Schwann cells in DRG explant cultures when combined with bFGF [ 185 ]. Interestingly, primary cultures of Schwann cells show density-dependent survival, due to these autocrine signaling circuits [ 56 , 78 , 83 , 164 ], and better survival in higher culture densities was mentioned for hiPSC-derived [ 118 ], as well as MSC-derived Schwann cells [ 186 ]. This suggests that autocrine survival, and possibly differentiation signals yet unknown, are also at play in these cultures.…”

“…A previous study, mentioned above, demonstrated that in the neural crest induction step, the performance of the same differentiation protocol can vary drastically between several hiPSC lines [ 142 ]. Indeed, work from our own lab, based on the protocol by Kim et al, also demonstrated a high variability across differentiation batches with hiPSC and showed that the efficiency and robustness of the neural crest induction step could be increased by additional activation of BMP signaling at a precisely controlled intermediate activity level [ 118 ] ( Table 2 ). A schematic overview of this modified protocol is depicted, alongside schematic expression timelines for some typical marker proteins, in Figure 4 .…”