Abstract
Regenerative capability of the peripheral nervous system after injury is enhanced by Schwann cells (SCs) producing several growth factors. The clinical use of SCs in nerve regeneration strategies is hindered by the necessity of removing a healthy nerve to obtain the therapeutic cells. Adipose-derived stem cells (ASCs) can be chemically differentiated towards a SC-like phenotype (dASCs), and represent a promising alternative to SCs. Their physiology can be further modulated pharmacologically by targeting receptors for neurotransmitters such as acetylcholine (ACh). In this study, we compare the ability of rat dASCs and native SCs to produce NGF in vitro. We also evaluate the ability of muscarinic receptors, in particular the M2 subtype, to modulate NGF production and maturation from the precursor (proNGF) to the mature (mNGF) form. For the first time, we demonstrate that dASCs produce higher basal levels of proNGF and mature NGF compared to SCs. Moreover, muscarinic receptor activation, and in particular M2 subtype stimulation, modulates NGF production and maturation in both SCs and dASCs. Indeed, both cell types express both proNGF A and B isoforms, as well as mNGF. After M2 receptor stimulation, proNGF-B (25 kDa), which is involved in apoptotic processes, is strongly reduced at transcript and protein level. Thus, we demonstrate that dASCs possess a stronger neurotrophic potential compared to SCs. ACh, via M2 muscarinic receptors, contributes to the modulation and maturation of NGF, improving the regenerative properties of dASCs.
Highlights
Nerve growth factor (NGF) has particular relevance for the survival and functional activity of sensory and sympathetic neurons[2,3]
In differentiation in SCs phenotype make Schwann–like (dASCs), M2 receptor activation produces a reversible decrease of cell proliferation, reduces migration and enhances dASCs differentiation as shown by improved spindle shaped morphology accompanied by early growth factor 2 (EGR2) upregulation33. dASCs produce neurotrophic factors, such as BDNF (Brain-derived neurotrophic factor, BDNF) and NGF, which are important for their neurotrophic effects as demonstrated in animal models of peripheral nerve regeneration[34,35]
Gene expression analysis showed that Arecaidine Propargyl Ester (APE) exposure was able to upregulate proNGF-A in dASCs compared to untreated controls (Fig. 1B), while a significant decrease was observed in Schwann cells (SCs) (Fig. 1E)
Summary
Nerve growth factor (NGF) has particular relevance for the survival and functional activity of sensory and sympathetic neurons[2,3]. We have evaluated the ability of muscarinic receptors to modulate NGF production and release in rat dASCs and SCs. For the first time, we demonstrate that dASCs produce and release higher levels of proNGF and mNGF than SCs. We have analysed the effects of non-selective muscarinic agonist stimulation (muscarine) and M2 selective agonist stimulation (APE) on NGF production and maturation in both dASCs and native SCs. Our results indicate that muscarinic receptor activation triggers NGF production both in SCs and in dASCs. Our results indicate that muscarinic receptor activation triggers NGF production both in SCs and in dASCs These results may contribute to define a new pharmacological target, improving the neurotrophic potential of dASCs towards new therapeutic approaches for peripheral nerve regeneration
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