Neurotrophic factor‐secreting autologous muscle stem cell therapy for the treatment of laryngeal denervation injury

Abstract

Objectives To determine if the spontaneous reinnervation that characteristically ensues after recurrent laryngeal nerve (RLN) injury could be selectively promoted and directed to certain laryngeal muscles with the use of neurotrophic factor (NF)-secreting muscle stem cell (MSC) vectors while antagonistic reinnervation is inhibited with vincristine (VNC). Study Design Basic science investigations involving primary cell cultures, gene cloning/transfer, and animal experiments. Methods (i.) MSC survival assays were used to test multiple individual NFs in vitro. (ii.) Motoneuron outgrowth assays assessed the trophic effects of identified NF on cranial nerve X-derived (CNX) motoneurons in vitro. (iii.) Therapeutic NF was cloned into a lentiviral vector, and MSCs were tranduced to secrete NF. 60 rats underwent left RLN transection injury, and at 3 weeks received injections of either MSCs (n=24), MSCs secreting NF (n=24), or saline (n=12) into the left thyroarytenoid muscle complex (TA); half of the animals in the MSC groups simultaneously received left posterior cricoarytenoid (PCA) injections of vincristine (VNC) while half the animals received saline. Results (i.) Ciliary-derived neurotrophic factor (CNTF) had the greatest survival-promoting effect on MSCs in culture. (ii.) Addition of CNTF (50 ng/mL) to CN X motoneuron cultures resulted in enhanced neurite outgrowth and branching. (iii.) In the animal model, the injected MSCs fused with the denervated myofibers, immunohistochemistry demonstrated enhanced reinnervation based on motor endplate to nerve contact, and RT-PCR confirmed stable CNTF expression at longest follow-up (4 months) in the CNTF-secreting MSC treated groups. Conclusions MSC therapy may have a future role in selectively promoting and directing laryngeal reinnervation after RLN injury. Level of evidence: NA

Autologous myoblasts attenuate atrophy and improve tongue force in a denervated tongue model: A pilot study

“…Our histologic findings of larger myofiber diameters in GFP+ myofibers support the hypothesis that MdSCs that fuse in the partially denervated ovine tongue have the ability to attenuate muscle atrophy. Similar effects have been noted in the rodent denervated thyroarytenoid muscle . The additional finding that muscle fiber size increased beyond preinjury levels may occur secondary to the MdSCs fusing with both normally innervated and denervated myofibers, and self‐fusing to form new myofibers.…”

Improved adductor function after canine recurrent laryngeal nerve injury and repair using muscle progenitor cells

“…The scale could be further tipped by inhibiting the antagonistic muscle from reinnervating, for example, by early injection of vincristine. We have previously reported that adductor function improved when reinnervation of the posterior cricoarytenoid was inhibited by injecting it with vincristine …”

“…Halum et al have successfully cultured MPCs from rat skeletal muscle and implanted them into denervated thyroarytenoid muscles. They found the injected MPCs survived in their new muscle, attenuated atrophy, fused with the native myofibers, and enhanced reinnervation when programmed to express neurotrophic factor . The strength of the MPC‐treated thyroarytenoid muscles could not be assessed in this rat model, however.…”

Persistence of plasmid‐mediated expression of transgenes in human mesenchymal stem cells depends primarily on CpG levels of both vector and transgene

“…Of particular biomedical interest is a safe genetic modification of mesenchymal stem cells (MSCs) for downstream clinical applications. It has been demonstrated that genetically modified MSCs survive better after transplantation and migrate and engraft more efficiently into cellular environment at the tissue damage sites . In addition, MSCs are considered as promising vehicles for a targeted delivery of therapeutic genes …”

“…However, a number of publications report on a fast (i.e. within a week or so) decline of expression of therapeutic genes by nonviral, primarily plasmid, constructs . At the cellular level, essential impact on plasmid‐mediated expression has a process of de novo methylation.…”