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

Abstract

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 study of nerve regeneration and myelination. The glial cells of the peripheral nervous system, Schwann cells (SC), are key facilitators of these processes; it is therefore crucial that the interactions of these cellular components are studied together. Direct contact between DRG neurons and glial cells provides additional stimuli sensed by specific membrane receptors, further improving the neuronal response. SC release growth factors and proteins in the culture medium, which enhance neuron survival and stimulate neurite sprouting and extension. However, SC require long proliferation time to be used for tissue engineering applications and the sacrifice of an healthy nerve for their sourcing. Adipose-derived stem cells (ASC) differentiated into SC phenotype are a valid alternative to SC for the set-up of a co-culture model with DRG neurons to study nerve regeneration. The present work presents a detailed and reproducible step-by-step protocol to harvest both DRG neurons and ASC from adult rats; to differentiate ASC towards a SC phenotype; and combines the two cell types in a direct co-culture system to investigate the interplay between neurons and SC in the peripheral nervous system. This tool has great potential in the optimization of tissue-engineered constructs for peripheral nerve repair.

Polymeric scaffolds for three-dimensional culture of nerve cells: a model of peripheral nerve regeneration

“…They also propose the use of their nanofibers for in vivo studies by seeding cells inside the scaffold, modifying it to a self-shaping fibrin tubular structure with the incorporation of a hydrogel in the electrospun fiber. [140],[165] …”

In vitro models for peripheral nerve regeneration

“…how closely they reproduce the effects of the natural nerve environment on Schwann cells or neurons (Ribeiro-Resende et al, 2009;Haastert-Talini et al, 2013); (iv) the specific changes in Schwann cell and/or neuronal behaviour upon single or multiple stimuli (e.g. trophic factor administration plus biostimulation by physical agents plus surface functionalization of biomaterials) (Brushart et al, 2013;van Neerven et al, 2013;Morano et al, 2014); and (v) the neuron-glial differentiation potential of stem cells (de Luca et al, 2015).…”

“…A dissociated DRG neuron culture can be co-cultured with different Schwann-like derived stem cells, providing a valuable model to study nerve regeneration and myelination in vitro, mimicking the in vivo environment at the injury site (de Luca et al, 2015).…”

In vitro evaluation of gel‐encapsulated adipose derived stem cells: Biochemical cues for in vivo peripheral nerve repair

“…, ). Compared with SC, adipose‐derived stem cells (ASC) can be harvested from white adipose tissue with traditional, non‐invasive liposuction techniques, and isolated through enzymatic and mechanical digestion procedures (Zuk et al ., ; Kingham, Mantovani, & Terenghi, ; de Luca, Faroni, & Reid, ). ASC present an elevated proliferation rate thereby reducing delays for cell transplantation (Gomillion & Burg, ); they prevent neuron apoptosis (Reid et al, ; Wei et al, ) and are characterized by a low immunological profile (Faroni, Smith, & Reid, ).…”

“…However, the beneficial role of transplanted SC at the site of injury of a peripheral nerve remains limited because SC atrophy over time, and their harvesting procedure is invasive and time-consuming (Gordon, Tyreman, & Raji, 2011;Hoke, 2006;Saheb-Al-Zamani et al, 2013). Compared with SC, adipose-derived stem cells (ASC) can be harvested from white adipose tissue with traditional, non-invasive liposuction techniques, and isolated through enzymatic and mechanical digestion procedures (Zuk et al, 2002;Kingham, Mantovani, & Terenghi, 2011;de Luca, Faroni, & Reid, 2015). ASC present an elevated proliferation rate thereby reducing delays for cell transplantation (Gomillion & Burg, 2006); they prevent neuron apoptosis (Reid et al, 2011;Wei et al, 2009) and are characterized by a low immunological profile (Faroni, Smith, & Reid, 2014).…”