Korean J Urol.  2008 Apr;49(4):350-359.

The Effects of Human Muscle Derived Stem Cells on the Induction of Peripheral Nerve Regeneration

• Koh JS
• Lee JY
• Lee JY

Abstract

PURPOSE: In this study, we evaluated the extent of functional and histological axonal regeneration after resection of the sciatic nerve. The nerve was repaired with silicone tubes filled with human muscle derived stem cells(MDSCs) and neuronal progenitor cells(NPCs) in nude mice.
MATERIALS AND METHODS
The human muscle samples were obtained from the rectus abdominis muscle of 12 patients that underwent a laparotomy. The MDSCs were isolated using a modified preplate technique. Using the MDSCs, neurogenic differentiation was induced by dissociating neurospheres produced in a neurosphere culture medium containing neuronal induction agents. A part of the sciatic nerve, approximately 7 mm in length, was excised bilaterally, and a 9mm long silicone tube guide was placed at the resulting gap in 40 nude mice. The transplanted sites were divided randomly into three groups according to the type of grafting cells: silicone tube guides filled with PBS(P group, n=20), silicone tube guides filled with MDSCs(M group, n=40) and silicone tube guides filled with NPCs(N group, n=20). Histological observations and a nerve conduction study were performed 12 weeks after the graft.
RESULTS
The number and diameter of the myelinated axons were significantly increased in the M and N groups(p<0.001). In a nerve conduction study, the amplitude of the compound muscle action potential(CMAP) and motor latency of response were significantly higher and shorter in the M and N groups(p<0.001). Moreover, reaction with neuronal class beta-tubulin(Tuj1, a neuronal marker) and antiglial fibrillary acidic protein(GFAP, a glial marker) was observed in the regenerated nerve that originated from the M and N groups.onclusions: These results show that MDSCs can differentiate into NPCs and improve the peripheral nerve regeneration rate after transplantation into a nerve guide.

Keyword

Muscles; Stem cells; Nude mouse; Nerve regeneration; Nerve conduction

MeSH Terms

Animals
Axons
Humans
Laparotomy
Mice
Mice, Nude
Muscles
Myelin Sheath
Nerve Regeneration
Neural Conduction
Neurons
Peripheral Nerves
Rectus Abdominis
Regeneration
Sciatic Nerve
Silicones
Stem Cells
Transplants
Silicones

Figure

• 

  Fig. 1 Pre and post neurogenic differentiation morphology of MDSCs grown as neurospheres. (A) Undifferentiated MDSCs: polygonal flat cells predominated, with a few cells with a round or triangular cell body. (B) MDSCs derived neurospheres were observed after 7 days in the neurospheres medium. (C, D) Morphology of cells isolated from neurospheres and plated on coverslips after 3 days. Bipolar, tripolar and large flat cells were observed. Scale bar=20 µm. MDSCs: muscle derived stem cells.

• 

  Fig. 2 Immunocytochemical staining. (A, C) Cells with a neuronal like morphology were positive for Tuj1 (white arrow). (B, D) Cells with a neuronal like morphology were positive for GFAP (white arrow). The nuclei are colored with DAPI. Tuj1: neuronal class III β-tubulin, GFAP: antiglial fibrillary acidic protein.

• 

  Fig. 3 Gross findings of regenerated tissue. (A) An empty tube filled with yellowish fluid was seen at 12 weeks after the grafting of PBS alone. (B) A regenerated nerve was seen in the tube at 12 weeks after the grafting of MDSCs. (C) A regenerated nerve was seen in the tube at 12 weeks after grafting of NPCs. PBS: phosphate buffered saline, MDSCs: muscle derived stem cells, NPCs: neuronal progenitor cells.

• 

  Fig. 4 Morphological analysis of axonal regeneration in the nude mouse sciatic nerve. (B, C, E, F) At 6 and 12 weeks, MDSCs and NPCs transplanted animals show numerous regenerated axons. Myelinated cells (black arrow) with a large diameter are observed. (G, H) Angiogenesis is remarkable around the nerve fibers. (A, D) There are no visible regenerated axons in the PBS filled silicone tube transplanted animals. Toluidine blue staining or H&E staining. Scale bar=100 µm. MDSCs: muscle derived stem cells, NPCs: neuronal progenitor cells, PBS: phosphate buffered saline.

• 

  Fig. 5 Comparison of the number (A) and diameter (B) of myelinated axons among the PBS, MSCs, and NPCs transplanted groups. Significant improvement of regeneration of myelinated axons was observed at 6 and 12 weeks after MDSCs and NPCs transplantation as compared with the infusion of PBS alone (*, †, ‡p<0.001). However, there were no significant differences in number and diameter of myelinated axons between the MSCs and NPCs transplantated groups. MDSCs: muscle derived stem cells, NPCs: neuronal progenitor cells, PBS: phosphate buffered saline.

• 

  Fig. 6 Immunohistochemical staining of regenerated tissue. Twelve weeks after transplantation immunofluorescence analysis showed an increased expression of Tuj1 (white arrow) and GFAP (white arrow) in the MDSCs and NPCs transplanted animals as compared to the PBS filled silicone tube transplanted animals. The nuclei are colored with DAPI. The existence of formerly transplanted stem cells was verified by the use of PKH dye (arrow head). Scale bar=50 µm. Tuj1: neuronal class III β-tubulin, GFAP: antiglial fibrillary acidic protein, MDSCs: muscle derived stem cells, NPCs: neuronal progenitor cells, PBS: phosphate buffered saline.

• 

  Fig. 7 At 12 weeks post-surgery, motor reinnervation was assessed by means of nerve conduction test. (A) No compound muscle action potentials (CMAPs) were measurable in any of the animals that received PBS alone. (B, C) Recovery of CMAPs appeared in both the MDSCs (B) and NPCs (C) transplanted groups. Comparison of the amplitude of CMAP (D) and motor latency of response (E) among the PBS, MDSCs, and NPCs transplanted groups. The amplitude of CMAP was significant higher in the animals that received MDSCs and NPCs transplantation (*p<0.001). The latency of response was significant shorter in the animals that received MDSCs and NPCs transplantation (†p<0.001). However, there were no significant differences in CMAPs and latency between the MDSCs and NPCs transplanted groups. CMAP: compound muscle action potentials, MDSCs: muscle derived stem cells, NPCs: neuronal progenitor cells, PBS: phosphate buffered saline.

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