Ex vivo expansion of skeletal muscle stem cells with a novel small compound inhibitor of eIF2α dephosphorylation

Abstract

BACKGROUND. Regeneration of adult tissues requires the activity of rare, mitotically quiescent somatic stem cells. These features are illustrated by the muscle stem cell (MuSC), also known as the satellite cell for its satellite position underneath the basal lamina of the myofiber.Isolation of MuSCs results in their rapid activation of the myogenic program and their subsequent culture ex vivo leads to loss of stem cell regenerative capacity. These shortcomings make MuSCs difficult to study, manipulate and prevent cell based therapies. We have previously shown that muscle stem cells (MuSCs) require tightly regulated protein synthesis through the phosphorylation of eIF2α. Sal003, an analog of salubrinal that blocks eIF2α dephosphorylation, promotes ex vivo expansion of MuSCs retaining regenerative capacity after engraftment into the Dmd mdx mouse model of Duchenne muscular dystrophy.METHODS. Since micromolar concentrations of sal003 (10μM) are required to expand MuSCs ex vivo, we undertook a structure relations study to identify novel sal003 analogs with efficacy at lower concentrations. We demonstrate ex vivo expansion of MuSCs isolated from wild-type and mdx mice using new compounds, and use CRISPR/Cas9 genome editing tools to restore dystrophin expression in cultured MuSCs.RESULTS. Here, we have synthesized and screened chemical analogs of sal003 to identify a novel compound promoting the ex vivo expansion of MuSCs. The novel compound expands wild-type and mdx MuSCs more efficiently than sal003 and also prolongs culture of primary myoblasts from isolated MuSCs.CONCLUSIONS. We identify a novel sal003 analog, C10, with increased potency at lower concentrations. Culture conditions including sal003 or C10 can extend culture of primary myoblasts from isolated MuSCs, which we predict will enable their further study, genetic manipulation and cell based therapies.

Towards stem cell therapies for skeletal muscle repair

“…Propagation with (R)-PFI-2 enhanced the therapeutic potential of MuSCs by restoring muscle strength in mdx mice after transplantation. Similarly, detailed studies by the Crist lab 16,17 have demonstrated the translational machinery of MuSCs can be targeted with chemical molecules to inhibit the production of pro-differentiation factors. Inhibitors of eIF2alpha phosphatase Gadd34/PP1 such as Sal003 17 as well as a more potent analog, 'C10' 16 , were shown to be particularly effective.…”

“…Similarly, detailed studies by the Crist lab 16,17 have demonstrated the translational machinery of MuSCs can be targeted with chemical molecules to inhibit the production of pro-differentiation factors. Inhibitors of eIF2alpha phosphatase Gadd34/PP1 such as Sal003 17 as well as a more potent analog, 'C10' 16 , were shown to be particularly effective. Treatment of mouse MuSCs with 5 µM C10 in culture media delayed activation of the myogenic program, boosted the expression of Pax7 and maintained the engraftment potential of MuSCs to levels comparable to the transplantation of freshly isolated cells.…”

“…Treatment of mouse MuSCs with 5 µM C10 in culture media delayed activation of the myogenic program, boosted the expression of Pax7 and maintained the engraftment potential of MuSCs to levels comparable to the transplantation of freshly isolated cells. Interestingly, C10 also permitted the expansion of MuSCs over multiple passages, generating up to 13 fold more Pax7+ MuSCs compared with standard culture conditions 16 . Together these studies demonstrate the utility of pharmacological inhibitors of myogenic differentiation to enhance propagation of MuSCs ex vivo and boosting transplantation potential.…”

Long-term high-yield skeletal muscle stem cell expansion through staged perturbation of cytokine signaling in a soft hydrogel culture platform

“…Reported expansion factors include the non-canonical Wnt7a ligand (Le Grand et al, 2009), the cyclic AMP activator forskolin (Xu et al, 2013), and pharmacological inhibitors of p38α/β mitogen-activated protein kinase (MAPK) (Bernet et al, 2014;Charville et al, 2015;Cosgrove et al, 2014), translational elongation factor eIF2-α (Lean et al, 2019;Zismanov et al, 2016), and the methyltransferase Setd7 (Judson et al, 2018). Furthermore, culture substrate engineering approaches have demonstrated that substrate biophysical parameters and extracellular matrix proteins can influence MuSC self-renewal ex vivo.…”

Satellite cell expansion is mediated by P-eIF2α dependentTacc3translation

“…Genetic inactivation of eIF2a phosphorylation (P-eIF2a) in satellite cells leads to increased global protein synthesis, activation of the myogenic program, and failure to self-renew. Pharmacological inhibition of eIF2a dephosphorylation with the small molecule sal003 (7,8)maintains low levels of protein synthesis and enables ex vivo expansion of cultured satellite cells that retain their regenerative potential (6,9).…”