Skin-derived precursors as a source of progenitors for cutaneous nerve regeneration

Abstract

Peripheral nerves have the potential to regenerate axons and reinnervate end organs. Chronic denervation and disturbed nerve regeneration are thought to contribute to peripheral neuropathy, pain, and pruritus in the skin. The capacity of denervated distal nerves to support axonal regeneration requires proliferation by Schwann cells, which guide regenerating axons to their denervated targets. However, adult peripheral nerve Schwann cells do not retain a growth-permissive phenotype, as is required to produce new glia. Therefore, it is believed that following injury, mature Schwann cells dedifferentiate to a progenitor/stem cell phenotype to promote axonal regrowth. In this study, we show that skin-derived precursors (SKPs), a recently identified neural crest-related stem cell population in the dermis of skin, are an alternative source of progenitors for cutaneous nerve regeneration. Using in vivo and in vitro three-dimensional cutaneous nerve regeneration models, we show that the SKPs are neurotropic toward injured nerves and that they have a full capacity to differentiate into Schwann cells and promote axon regeneration. The identification of SKPs as a physiologic source of progenitors for cutaneous nerve regeneration in the skin, where SKPs physiologically reside, has important implications for understanding early cellular events in peripheral nerve regeneration. It also provides fertile ground for the elucidation of intrinsic and extrinsic factors within the nerve microenvironment that likely play essential roles in cutaneous nerve homeostasis.

Figure 1. Implanted SKPs are Neurotropic In Vitro and In Vivo

1A. In Vitro Engineered Skin Raft for gliogenesis/nerve regeneration. Inserts of six-well tissue culture plates were coated with 1 ml bovine collagen I and layered with 3 ml collagen I containing 1×10⁵ human foreskin fibroblasts. After 7 days of incubation at 37°C, keratinocytes were seeded on top of the dermal rafts. The skin equivalent rafts were kept submerged in medium for 2 days and then raised to the air-liquid interface via feeding with medium bellow. SKPs and DRGs/nerves were introduced into the skin constructs by mixing with fibroblasts and then adding them to the dermal fibroblast/collagen layer. Extracellular factors essential for this process, including neuregulin, other Schwann cell factors, and/or nerve growth factors were added to the media. 1B. X-gal staining of an in vitro engineered skin raft containing only fibroblasts, DRGs/nerves (a) or only fibroblasts and lacZ positive SKPs (b). In skin rafts that contain lacZ positive SKPs, fibroblasts and DRGs/nerves, the Lacz positive SKPs concentrate near the DRGs/nerves (c–d; arrows). Paraffin sections of these DRGs in (c) and (d) showing the LacZ positive SKPs infiltrating into the DRGs, forming cellular corridors, similar to the bands of Bungner (e, arrows) as well as differentiating into LacZ-postive Schwann-like cells that wrap around axons within the DRGs (f, arrows). Scale bar = 20 μm. 1C. SKPs were harvested from skin from the back & neck of CMV-CreER^T2;Rosa26 mice and exposed to 4-OH-tamoxifen to induce recombination at the Rosa 26 locus and then reimplanted dermally in the same mice at the dorsal/sacral area (a). Grafted sites were harvested two months later for cutaneous nerve analysis. X-gal staining shows that blue lacZ positive SKPs migrate to and reside predominantly within the cutaneous nerves (b–c, arrows). Scale bar = 20 μm.

Figure 2. Nerve microenvironment promotes the differentiation of SKPs into Schwann cells

In vitro engineered skin rafts containing lacZ-postive SKPs, fibroblasts and DRGs/nerves were harvested after 4–6 weeks in culture, using X-gal staining to trace the location of the SKPs. Tissues were then processed for histological and immunohistochemical analysis. The majority of blue cells (marker for SKPs) are also positive for GAP 43 (a Schwann cell marker) within and in the vicinity of the DRGs/nerves (A–D). There are tubal structures or cellular corridors of SKP-derived GAP 43 positive Schwann cells in these areas (C, arrows). In contrast, there are only a few LacZ-GAP 43 double positive cells in areas distant from the DRGs and nerves (E, F and arrows). Scale bar = 20 μm.

Figure 3. SKPs contributed to peripheral nerve regeneration

SKPs were harvested from skin on the back & neck of CMV-CreER^T2;Rosa26 mice and exposed to 4-OH-tamoxifen to induce recombination at the Rosa 26 locus and then reimplanted back to the same animals, adjacent to the sciatic nerve (A). X-gal staining demonstrates a network of LacZ-positive nerves wrapping around the sciatic nerve where SKPs were implanted (B). Immunohistochemical analysis with GAP43, a Schwann cell marker (C, brown arrows) and Holmes stain, a neurofibril/axon marker (D–E, black arrows) establish that the LacZ-positive bundles and branches (blue arrows) are nerve fibers. Scale bar = 20 μm.

Figure 4. SKPs as a source of autologous progenitors for cutaneous nerve regeneration

Cutaneous nerve injuries were generated with axonal transections within the dermis by complete excision of a 1.5 cm circular island of skin on the back. The excised skin was then grafted autologously back into the same position, covered with sterile petrolatum gauze and secured with sutures. LacZ-positive SKPs harvested from the same animal were implanted into the graft and the wounds allowed to heal for cutaneous nerve regeneration (A–a). Immunohistochemical analysis with GAP43 showed that SKPs do not contain Schwann cells prior to transplantation as they are GAP43 negative (A–b,c,d). X-gal staining of the excised skin graft 4–6 weeks after implantation demonstrates a network of LacZ-positive new nerves sprouting out of the proximal stumps (dotted lines) into the graft where the SKPs were implanted (B, arrows). Holmes stain, a neurofibril/axon marker (C) and immunohistochemical analysis with GAP43, a Schwann cell marker (D–E) establish that these LacZ-positive branches (arrows) are nerve fibers. Scale bar = 20 μm.

Figure 5. SKPs can serve as a source of allogeneic progenitors for cutaneous nerve regeneration

Cutaneous nerve injuries were made in athymic mice (BALB/c background) with axonal transections within the dermis through excision of a 1.5 cm circular island of skin on the back. The excised skin was then grafted autologously back into the same position. The LacZ-positive SKPs harvested from different mice (C57BL/6 background) were implanted into the graft and the wounds allowed to heal for cutaneous nerve regeneration (A). X-gal staining of the excised skin graft 4–6 weeks after implantation demonstrates LacZ-positive cutanous nerves in the grafts, where the SKPs were implanted (B, arrows). Holmes stain, a neurofibril/axon marker (C) and immunohistochemical analysis with GAP43, a Schwann cell marker (D–E) and MBP (F) establish that the LacZ-positive branches (arrows) are myelinating nerve fibers. Scale bar = 20 μm.

Figure 6. Physiologic and pathophysiologic roles of SKPs in the skin

SKPs are multipotent neural crest stem cell (NCSC)-related precursors in the skin that may play important roles in cutaneous nerve homeostasis. Under physiologic conditions, cutaneous nerve transection may recruit neighboring SKPs into the injured sites and promote the differentiation of SKPs into Schwann cells. The proliferating Schwann cells also secrete chemoattractant factors that recruit other cell types, including fibroblasts that undergo cell sorting, where Schwann cells and fibroblasts grow in ordered columns along the endoneurial tube creating cellular corridors, known as “bands of Bungner”, to guide regenerating axons across the injured gaps [2, 3]. On the other hand, under NF1 pathologic condition, neurons/trauma may produce “physiologic factors” that preferentially induce SKPs to differentiate toward the Schwann cell lineage. During Schwann cell differentiation, Loss of Heterozygosity of NF1 expression in the SKPs or early Schwann cell differentiation, in addition to other microenvironmental cues (such as hormones, neurons, inflammation, etc.), leads to neurofibroma formation [29]. This is in alignment with the human clinical scenario, because it is known that trauma to the skin of NF1 patients can induce dermal neurofibroma formation [56].