Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An <em>In Vitro</em> Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the ...

The University of Manchester

We investigated the effect of cobalamin (Cbl) deficiency on epidermal growth factor receptor (EGFR) mRNA levels in the spinal cord (SC) and liver of rats made Cbl-deficient (Cbl-D) by means of total gastrectomy or a Cbl-D diet, and simultaneously measured the levels of the epidermal growth factor (EGF). Both methods of inducing Cbl deficiency decreased EGFR expression in the SC and liver. Cbl replacement treatment normalized or nearly so most of the abnormalities in EGFR expression in the totally gastrectomized (TGX) rats at different times. The EGFR-immunostaining intensity decreased in the SC white matter of the Cbl-D rats and significantly increased in that of the TGX, Cbl-treated rats. EGF levels significantly increased in liver of TGX rats and in SC of 4-month TGX rats, and the increases returned to almost normal levels after a postoperative 2-month administration of Cbl to TGX rats. These findings demonstrate that Cbl deficiency dysregulates the EGFR-EGF dyad in these tissues.

Cobalamin deficiency-induced changes of epidermal growth factor (EGF)-receptor expression and EGF levels in rat spinal cord.

γ-Aminobutyric acid type B receptors (GABA-B) are expressed in glial cells of the central and peripheral nervous systems, and recent evidence has shown their importance in modulating physiological parameters of Schwann cell (SC). SC play essential roles in peripheral nerve regeneration, but several drawbacks prevent their use for nerve repair. Adult stem cells from adipose tissue (ASC) or bone marrow (BM-MSC) can be differentiated into an SC-like phenotype and used as SC replacements. The aim of this study was to investigate GABA-B receptor functional expression in differentiated stem cells by assessing the similarity to SC. By means of RT-PCR and Western blot methodologies, BM-MSC and ASC were found to express both GABA-B1 and GABA-B2 receptor subunits. The expression levels of GABA-B1b and GABA-B2 receptors were influenced by SC-like differentiation, as shown by Western blot studies. GABA-B receptor stimulation with baclofen reduced the proliferation rate of SC and differentiated ASC (dASC) but not that of dBM-MSC. In conclusion, both of the subunits that assemble into a functional GABA-B receptor are present in differentiated stem cells. Furthermore, GABA-B receptors in dASC are functionally active, regulating a key process such as proliferation. The presence of functional GABA-B receptors on differentiated stem cells opens new opportunities for a possible pharmacological modulation of their physiology and phenotype.

Schwann-like adult stem cells derived from bone marrow and adipose tissue express γ-aminobutyric acid type B receptors.

γ-Aminobutyric acid (GABA) receptors are present in peripheral and central glia and modulate important physiological parameters of glial cells. Schwann cells (SC), the peripheral nervous system glial cells, play essential roles in nerve regeneration, but they are unsuitable for bioengineering of nerve repair. Increasing interest has been focused on adult stem cells derived from bone marrow (BM-MSC) or adipose tissue (ASC), which can be differentiated into SC-like phenotype and used as SC replacements. SC-like adult stem cells express GABA-B receptors that can modulate their proliferation. The aim of this study was to investigate GABA-A receptors functional expression in differentiated stem cells. BM-MSC and ASC were found to express GABA-A α2 and β3, but not β1 mRNA transcripts. Protein expression levels of GABA-A α2 and β3 receptors were upregulated following SC-like differentiation as shown by Western blot studies. GABA-A receptor stimulation with muscimol increased the proliferation rate of SC, differentiated BM-MSC and differentiated ASC. In conclusion, GABA-A α2 and β3 receptor subunits are present in BM-MSC and ASC and upregulated following glial differentiation. GABA-A subunits in differentiated stem cells and SC assemble in functional receptors modulating cell proliferation. Functional GABA-A and GABA-B receptors represent a possible pharmacological target to modulate SC-like stem cells physiology.

Expression of functional γ-aminobutyric acid type A receptors in Schwann-like adult stem cells.

Neurotrophins are a group of polypeptides that specifically influence neuronal activity during development and adult life in the central and peripheral nervous system (PNS). In particular, Schwann cells (SC) in the PNS exert a neurotrophic role following up-regulation of several growth factors, including nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF). Also SC-like cells derived from adipose tissue (dASC), which have molecular and functional properties similar to SC, can produce and secrete NGF and BDNF. Interestingly, gamma-aminobutyric acid (GABA) and its receptors have been also suggested as modulators of development and myelination in PNS. Therefore, it was interesting to investigate whether the stimulation of the GABA-B receptor may regulate the expression of neurotrophins in SC and dASC. Our findings demonstrated that the specific GABA-B receptors agonist baclofen influences the expression and the secretion NGF and BDNF. In particular, 2 and 24 h of baclofen exposure lead to increased neurotrophins expression in both SC and dASC, as measured by western blot. Moreover, enzyme-linked immunosorbent assay showed that also the levels of released neurotrophins were modified after baclofen treatments. The possibility to modulate the neurotrophic potential of adult stem cell, acting on functional GABAergic receptors, could represent a novel pharmacological approach to improve nerve regeneration.

Baclofen modulates the expression and release of neurotrophins in schwann-like adipose stem cells.

Since the first observations on the existence of "neurosteroids" in the 1980s, our understanding of the importance of these endogenous steroids in the control of the central and peripheral nervous system (PNS) has increased progressively. Although most of the observations were made in neuronal cells, equally important are the effects that neurosteroids exert on glial cells. Among the different classes of neurosteroids acting on glial cells, the progesterone 5α-3α metabolite, allopregnanolone, displays a particular mechanism of action involving primarily the modulation of classic GABA receptors. In this review, we focus our attention on allopregnanolone because its effects on the physiology of glial cells of the central and PNS are intriguing and could potentially lead to the development of new strategies for neuroprotection and/or regeneration of injured nervous tissues.

The neurosteroid allopregnanolone modulates specific functions in central and peripheral glial cells.

The gold standard in surgical management of a peripheral nerve gap is currently autologous nerve grafting. This confers patient morbidity and increases surgical time therefore innovative experimental strategies towards engineering a synthetic nerve conduit are welcome. We have developed a novel synthetic conduit made of poly ε-caprolactone (PCL) that has demonstrated promising peripheral nerve regeneration in short-term studies. This material has been engineered to permit translation into clinical practice and here we demonstrate that histological outcomes in a long-term in vivo experiment are comparable with that of autologous nerve grafting. A 1cm nerve gap in a rat sciatic nerve injury model was repaired with a PCL nerve conduit or an autologous nerve graft. At 18 weeks post surgical repair, there was a similar volume of regenerating axons within the nerve autograft and PCL conduit repair groups, and similar numbers of myelinated axons in the distal stump of both groups. Furthermore, there was evidence of comparable re-innervation of end organ muscle and skin with the only significant difference the lower wet weight of the muscle from the PCL conduit nerve repair group. This study stimulates further work on the potential use of this synthetic biodegradable PCL nerve conduit in a clinical setting.

Long term peripheral nerve regeneration using a novel PCL nerve conduit.

In order to improve the outcome of nerve regeneration following peripheral trauma injuries, the development of bioengineered nerve grafts has attracted great attention in the field of tissue engineering. Adult stem cells constitute the ideal alternative to Schwann cells (SCs) as transplantable cells in bioartificial nerve grafts. Among the various sources of stem cells with potential applications for regenerative medicine, the adipose tissue has been proven to be one of the most promising. Adipose-derived stem cells (ASCs) are easily obtained, rapidly expanded, show low immunogenicity, and can be differentiated into SCs in vitro. This chapter will focus on recent advances in the use of differentiated and undifferentiated ASCs for peripheral nerve regeneration, with a critical attention for the clinical exploitability of ASC in nerve repair strategies.

Adipose-derived stem cells and nerve regeneration: promises and pitfalls.

The mechanisms regulating the differentiation into non-myelinating Schwann cells is not completely understood. Recent evidence indicates that GABA-B receptors may regulate myelination and nociception in the peripheral nervous system. GABA-B receptor total knock-out mice exhibit morphological and molecular changes in peripheral myelin. The number of small myelinated fibers is higher and associated with altered pain sensitivity. Herein, we analyzed whether these changes may be produced by a specific deletion of GABA-B receptors in Schwann cells. The conditional mice (P0-GABA-B1(fl/fl)) show a morphological phenotype characterized by a peculiar increase in the number of small unmyelinated fibers and Remak bundles, including nociceptive C-fibers. The P0-GABA-B1(fl/fl) mice are hyperalgesic and allodynic. In these mice, the morphological and behavioral changes are associated with a downregulation of neuregulin 1 expression in nerves. Our findings suggest that the altered pain sensitivity derives from a Schwann cell-specific loss of GABA-B receptor functions, pointing to a role for GABA-B receptors in the regulation of Schwann cell maturation towards the non-myelinating phenotype.

Deletion of GABA-B receptor in Schwann cells regulates remak bundles and small nociceptive C-fibers.

Neuropathic pain arises as a direct consequence of a lesion or disease affecting the peripheral somatosensory system. It may be associated with allodynia and increased pain sensitivity. Few studies correlated neuropathic pain with nerve morphology and myelin proteins expression. Our aim was to test if neuropathic pain is related to nerve degeneration, speculating whether the modulation of peripheral GABA-B receptors may promote nerve regeneration and decrease neuropathic pain. We used the partial sciatic ligation- (PSL-) induced neuropathic model. The biochemical, morphological, and behavioural outcomes of sciatic nerve were analysed following GABA-B ligands treatments. Simultaneous 7-days coadministration of baclofen (10 mg/kg) and CGP56433 (3 mg/kg) alters tactile hypersensitivity. Concomitantly, specific changes of peripheral nerve morphology, nerve structure, and myelin proteins (P0 and PMP22) expression were observed. Nerve macrophage recruitment decreased and step coordination was improved. The PSL-induced changes in nociception correlate with altered nerve morphology and myelin protein expression. Peripheral synergic effects, via GABA-B receptor activation, promote nerve regeneration and likely ameliorate neuropathic pain.

Nerve regenerative effects of GABA-B ligands in a model of neuropathic pain.

Injuries to peripheral nerves are common and cause life-changing problems for patients alongside high social and health care costs for society. Current clinical treatment of peripheral nerve injuries predominantly relies on sacrificing a section of nerve from elsewhere in the body to provide a graft at the injury site. Much work has been done to develop a bioengineered nerve graft, precluding sacrifice of a functional nerve. Stem cells are prime candidates as accelerators of regeneration in these nerve grafts. This review examines the potential of adipose-derived stem cells to improve nerve repair assisted by bioengineered nerve grafts.

Adipose derived stem cells and nerve regeneration.

We have modified the surface topography of poly ɛ-caprolactone (PCL) and polylactic acid (PLA) blended films to improve cell proliferation and to guide the regeneration of peripheral nerves. Films with differing shaped grooves were made using patterned silicon templates, sloped walls (SL), V-shaped (V), and square-shaped (SQ), and compared with nongrooved surfaces with micropits. The solvent cast films were tested in vitro using adult adipose-derived stem cells differentiated to Schwann cell-like cells. Cell attachment, proliferation, and cell orientation were all improved on the grooved surfaces, with SL grooves giving the best results. We present in vivo data on Sprague-Dawley rat sciatic nerve injury with a 10-mm gap, evaluating nerve regeneration at 3 weeks across a polymer nerve conduit modified with intraluminal grooves (SL, V, and SQ) and differing wall thicknesses (70, 100, 120, and 210 μm). The SL-grooved nerve conduit showed a significant improvement over the other topographical-shaped grooves, while increasing the conduit wall thickness saw no positive effect on the biological response of the regenerating nerve. Furthermore, the preferred SL-grooved conduit (C) with 70 μm wall thickness was compared with the current clinical gold standard of autologous nerve graft (Ag) in the rat 10-mm sciatic nerve gap model. At 3 weeks postsurgery, all nerve gaps across both groups were bridged with regenerated nerve fibers. At 16 weeks, features of regenerated axons were comparable between the autograft (Ag) and conduit (C) groups. End organ assessments of muscle weight, electromyography, and skin reinnervation were also similar between the groups. The comparable experimental outcome between conduit and autograft, suggests that the PCL/PLA conduit with inner lumen microstructured grooves could be used as a potential alternative treatment for peripheral nerve repair.

Polymer Scaffolds with Preferential Parallel Grooves Enhance Nerve Regeneration.

Peripheral nerve injuries represent a substantial clinical problem with insufficient or unsatisfactory treatment options. This review summarises all the events occurring after nerve damage at the level of the cell body, the site of injury and the target organ. Various experimental strategies to improve neuronal survival, axonal regeneration and target reinnervation are described including pharmacological approaches and cell-based therapies. Given the complexity of nerve regeneration, further studies are needed to address the biology of nerve injury, to improve the interaction with implantable scaffolds, and to implement cell-based therapies in nerve tissue engineering.

Alessandro Faroni

Blond McIndoe Research Laboratories,
Institute of Inflammation & Repair,
Blond McIndoe Research Laboratories, Institute of Inflammation & Repair

The University of Manchester

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Alessandro Faroni

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The University of Manchester

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Blond McIndoe Research Laboratories,
Institute of Inflammation & Repair,
Blond McIndoe Research Laboratories, Institute of Inflammation & Repair