Implanted hair follicle stem cells form Schwann cells that support repair of severed peripheral nerves

Abstract

The hair follicle bulge area is an abundant, easily accessible source of actively growing, pluripotent adult stem cells. Nestin, a protein marker for neural stem cells, also is expressed in follicle stem cells and their immediate, differentiated progeny. The fluorescent protein GFP, whose expression is driven by the nestin regulatory element in transgenic mice, served to mark the follicle cell fate. The pluripotent nestin-driven GFP stem cells are positive for the stem cell marker CD34 but negative for keratinocyte marker keratin 15, suggesting their relatively undifferentiated state. These cells can differentiate into neurons, glia, keratinocytes, smooth muscle cells, and melanocytes in vitro. In vivo studies show the nestin-driven GFP hair follicle stem cells can differentiate into blood vessels and neural tissue after transplantation to the subcutis of nude mice. Equivalent hair follicle stem cells derived from transgenic mice with beta-actin-driven GFP implanted into the gap region of a severed sciatic nerve greatly enhance the rate of nerve regeneration and the restoration of nerve function. The follicle cells transdifferentiate largely into Schwann cells, which are known to support neuron regrowth. Function of the rejoined sciatic nerve was measured by contraction of the gastrocnemius muscle upon electrical stimulation. After severing the tibial nerve and subsequent transplantation of hair follicle stem cells, walking print length and intermediate toe spread significantly recovered, indicating that the transplanted mice recovered the ability to walk normally. These results suggest that hair follicle stem cells provide an important, accessible, autologous source of adult stem cells for regenerative medicine.

Fig. 1.

Rejoining severed sciatic nerve with hair follicle stem cells. (a1) Schematic of vibrissa follicle of GFP transgenic mice showing the position of GFP- and nestin-expressing vibrissa follicle bulge area (red arrowheads). (a2) Colony formed from GFP-expressing hair follicle stem cells from the vibrissa after 2 months in culture. (a3) GFP-expressing cells within the colony were nestin-positive. (b) GFP-expressing hair follicle stem cells grown for two months in DMEM-F12 containing B-27, 1% methylcellulose, and basic FGF were transplanted between the severed sciatic nerve fragments in C57BL/6 immunocompetent mice (white arrowheads). (c1 and c2) Fluorescence images from a live mouse. Two months after transplantation between the severed sciatic nerve, the GFP-expressing cells joined the severed sciatic nerve. c2 shows higher magnification of c1. (d1 and d2) Brightfield (d1) and fluorescence (d2) images of an excised sciatic nerve. The preexisting sciatic nerve is denoted by white arrowheads.

Fig. 2.

Cell types growing in area of sciatic nerve joined by hair follicle stem cells. (a) GFP-expressing vibrissa hair follicle stem cells were growing in the joined sciatic nerve. Most of the GFP-expressing vibrissa hair follicle stem cells differentiated to Schwann cells and formed myelin sheaths surrounding axons (red arrowheads). The axons are denoted by black arrowheads. (b) Transverse section of joined nerve. In the central area of the joined nerve, GFP-expressing cells formed many small myelin sheaths (white arrowheads). (c1) In the marginal area of the joined nerve, GFP-expressing cells formed many myelin sheaths (white arrowheads). (c2) Higher magnification of area of c1 indicated by the white dashed box.

Fig. 3.

Cell types growing in joined part of sciatic nerve. (a1-a3) GFP-expressing hair follicle stem cells differentiated to glial fibrillary acidic protein-positive Schwann cells after injection between the fragments of the severed sciatic nerve (white arrowheads). (b1-b3) GFP-expressing Schwann cells formed myelin sheaths and surrounded β-III-tubulin-positive axons (white arrowheads).

Fig. 4.

Electrical stimulation of rejoined sciatic nerve. (a) Four weeks after transplantation of GFP-expressing hair follicle stem cells between the severed sciatic nerve fragments, the rejoined sciatic nerve contracted the gastrocnemius muscle upon electrical stimulation. The sciatic nerve was stimulated above where the nerve was severed (white dashed area). (a1) Brightfield and fluorescence. (a2) Fluorescence. (b1and b2) Electrical stimulation of the sciatic nerve above where the nerve was severed after the nerve was rejoined by hair follicle stem cells. (Left) Before electrical stimulation. (Right) After electrical stimulation. (b3) Comparison of the extent of gastrocnemius muscle contraction in transplanted and untransplanted control mice. ^**, P < 0.01 vs. control.

Fig. 5.

Walking track analysis of GFP-expressing hair follicle stem cell transplantation in the tibial transection. Walking tracks were prepared as previously described using a 6- × 44-cm corridor open at one end to a darkened compartment. The tracks were evaluated for print length and intermediate toe spread. (a) After 6, 9, and 12 weeks, stem cell transplantation enabled the walking print length factor to recover as compared with untransplanted controls. (b) After 6, 9, and 12 weeks, stem cell transplantation enabled the intermediate toe spread factors to recover as compared with untransplanted controls. ^*, P < 0.05; ^**, P < 0.01 vs. control (without transplantation).