|SKPing towards Nerve Regeneration|
Original article from STEM CELLS
Studies have taught us that while peripheral cutaneous nerves have an amazing regenerative capability in response to damage (Navarro et al), numerous human diseases are associated with incomplete nerve regeneration. Proliferating Schwann cells are key players in nerve regeneration by guiding axons towards their denervated targets (Webber and Zochodne) and secreting neurotrophins to support injured axons and prevent apoptosis (Tofaris et al). However, these Schwann cells lack the sustained growth ability needed for the production of glial cells; the non-neuronal cells that maintain homeostasis, form myelin, and provide support and protection for neurons. It had been hypothesised that mature Schwann cells dedifferentiated into progenitor/stem-like cells to promote axonal regrowth following injury (Scherer and Salzer). However, the discovery of neural crest stem cells at sites of gliogenesis in the peripheral nervous system and neural crest-like stem cells in the skin (see original paper for references) suggests that resident adult stem cells may be the source of progenitors for cutaneous nerve regeneration. Two groups have published reports on the identification of skin-derived precursors/progenitors (SKPs); self-renewing, multipotent neural stem/precursor cells which are distinct from other known stem/precursor cells within the skin, which have the capacity to differentiate along neuronal and glial lineages (Toma et al, McKenzie et al and Li et al) and into glial cells in response to the nerve microenvironment within spinal cord and sciatic nerve injuries in mouse models (McKenzie et al, Biernaskie et al and Walsh et al). Now, in a study published in Stem Cells researchers from the laboratory of Lu Q. Le demonstrate that SKPs are a potential source of progenitors for cutaneous nerve regeneration and further show the critical role SKPs play in cutaneous nerve homeostasis through the use of in vivo and in vitro three-dimensional cutaneous nerve regeneration models (Chen and Pradhan et al).
A 3D in vitro model consisting of layers of collagen, human foreskin fibroblasts and keratinocytes were used for histological and immunohistochemical analysis. To faithfully mimic the in vivo situation, cellular components, such as SKPs, Schwann cells, neurons and dorsal root ganglions (DRGs) with associated nerves could be mixed with the fibroblast layer, while extracellular factors could be added to the culture medium. The use of a lacZ reporter mouse allowed for the identification of SKPs through X-gal staining, which demonstrated an even spread of SKPs in a 3D culture of SKPs and fibroblasts. However upon the addition of DRGs/associated nerves SKPs were not evenly spread, being found near these DRGs/associated nerves suggesting that SKPs exhibit neurotropic behaviour and migrate toward nerves. Deeper analysis found that SKPs had infiltrated the DRGs and some had differentiated into myelinating Schwann-like cells that ensheathed axons within the DRGs. To analyse SKPs behaviour in vivo, skin from CMV-CreERT2;Rosa26 mice was harvested, treated with 4-OHT to induce recombination at the Rosa 26 loci to generate LacZ+ SKPs which were then dermally transplanted into the same mice from which the SKPs has been harvested. 2 months after grafting, lacZ+ cells were again found predominantly associated with cutaneous nerves; overall suggesting that nerve microenvironment attracts or enhances SKP migration and survival.
SKP fate lineage was next investigated using the 3D model with lacZ+ SKPs, fibroblasts, and DRGs/associated nerves, and revealed that 6-8 weeks after construction and incubation, lacZ+ cells were found near to or within DRGS/associated nerves which were also positive for the Schwann cell marker GAP43. Interestingly, only a few lacZ+GAP43+ cells were not associated with DRGS/associated nerves suggesting that, when the microenvironment is right, the SKPs differentiate into Schwann cells and can contribute to peripheral gliogenesis. To confirm this, the mouse graft model was again used, but this time SKPs were re-implanted into sciatic nerves allowing for close proximity to a large peripheral nerve. At two months the SKPs had given rise to new nerve bundles and branches which were lacZ+GAP43+ confirming that in a favourable microenvironment SKPs can give rise to peripheral nerves.
Lastly, the ability of SKPs to aid in cutaneous nerve regeneration/gliogenesis was assayed. This involved the excision of a 1.5cm circle of epidermis from the back of a C57BL6 mouse and replaced in the same position, so generating many axonal transections. A pure population of lacZ+ SKPs was then implanted into the graft and analysed after healing. Excitingly, this demonstrated that new sprouting nerves were lacZ+, GAP43+ and also myelin basic protein (MBP)+ suggesting that these were SKP-derived myelinating nerve fibres.
Overall this exciting piece of research strongly suggests that SKPs are neurotropic in vivo and in vitro, they have the potential to contribute towards peripheral gliogenesis, they differentiate into Schwann cells and, excitingly, participate in the regeneration of injured cutaneous nerves. The future study of SKPs should allow us to further our understanding of normal cutaneous nerve homeostasis and also allow us to use this knowledge to address associated diseases. Perhaps the next step is to understand whether these cells can be propagated and amplified in vivo, whether this has any detrimental effects and if this research can be translated to humans.
Biernaskie J et al.
Chen, Z and Pradhan, S et al.
Li L et al.
McKenzie IA et al.
Navarro X et al.
Scherer SS, and Salzer, J.L.
Tofaris GK et al.
Toma JG et al.
Walsh SK et al.
Webber C, Zochodne D.
STEM CELLS correspondent Stuart P Atkinson reports on those studies appearing in current journals that are destined to make an impact on stem cell research and clinical studies.