Quiescent pluripotent stem cells reside within murine peripheral nerves that can be stimulated to proliferate by recombinant human bone morphogenic protein 2 or by nerve trauma

Abstract

BACKGROUND: The clinical use of recombinant human bone morphogenic protein 2 (rhBMP2, Infuse) has been associated with nerve-related complications including new-onset sciatica, and retrograde ejaculation.

PURPOSE: To better understand the interaction of rhBMP-2 and peripheral nerves with the intent of making procedures safer.

STUDY DESIGN/SETTING: Using a mouse model to examine the direct effect of diluted rhBMP-2 (Infuse) on murine sciatic nerves.

METHODS: Animal studies were approved by the Institutional Animal Care and Use Committee. Balb/c mouse sciatic nerves were surgically exposed and 60 ng (in 10 mu L) of rhBMP-2 was applied to the nerve. In separate experiments, the sciatic nerves were subjected to mechanical compression using forceps (and not exposed to rhBMP-2). The third group of mice received direct injection of the same amount of rhBMP-2, or sterile saline as a control, into the hamstring area of the posterior thigh without surgery. Mouse limbs with intact sciatic nerve were collected at 24, 48, or 72 hours after treatment for histology processing. A separate set of identically treated sciatic nerves were retrieved from mice at the same time points and cells were isolated by collagenase and trypsin digestion. The isolated cells were cultured in a stem cell medium containing 20% knockout serum and human leukemia inhibitory factor. Immunohistochemical or immunofluorescent cell stains against KLF4, Sox2, c-Myc, and Oct4 were performed on the mouse tissue sections and cell culture slides. In addition, real-time polymerase chain reaction (PCR) was performed to quantify the mRNA expression profiles of the stem cell marker genes in cultured cells.

RESULTS: Profound morphological changes of the mouse sciatic nerves were noted after exposure to rhBMP-2, with a rapid and robust cell proliferation within the nerves followed by migration of these cells into surrounding tissue. Immunohistochemical stain revealed strong nuclear stains of KLF4, Sox2, Oct4, and c-Myc on the overwhelming majority of these proliferating cells in the nerve. Intramuscular injections of rhBMP-2 or willful physical compression of the nerves showed similar cell proliferation effects as the direct application of Infuse to the sciatic nerve. The cells in stem cell culture medium grew steadily without feeder cells and appeared fairly uniform. They were adherent to substrate and were motile. Double fluorescent staining on the cells indicated colocalization of all pairs of the four stem cell markers in the cell nuclei. Real-time PCR confirmed the strong mRNA expressions of KLF4, Sox2, Oct4, and c-Myc in these isolated cells.

CONCLUSION: Exposure to BMP-2 causes a marked proliferation of previously quiescent cells within peripheral nerves. These cells simultaneously express KLF4, Sox2, Oct4, and c-Myc, the transcription factors that confer embryonic pluripotency. Work described in the companion paper reveals some of the differentiation capacity of the cells and their likely clinical significance. In addition, the effects of direct exposure of nerves to rhBMP-2 as described here should clearly illuminate the mechanism of BMP-2-related nerve complications. We would suggest that the use of this agent in proximity to known neural structures should only be done with extreme caution.