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In Vivo Tracking Of Amniotic Fluid Derived Stem Cells on Acellular Nerve Graft
Xue Ma, MD, PhD; Tianyi David Luo, MD; Zhongyu Li, MD, PhD; Thomas L. Smith, PhD
Wake Forest University, Winston Salem, NC

Introduction: Traumatic transections of peripheral nerves are associated with poor nerve regeneration. The use of nerve grafts with stem cells provides an alternative to autograft for nerve repair. The purpose of this study was to track the fate of amniotic fluid derived stem (AFS) cells that are seeded into nerve allografts and to elucidate the mechanism of their impact on the regenerating nerve.

Methods: AFS cells were labeled using supraparamagnetic micron sized iron oxide (MPIO) coated with fluorescent dye. Labeled cells were plated and viability was assessed. Next, cells were cultured in neurogenic induction media; the conditioned media was collected to evaluate the neurogenic growth factors. Differentiated cells were confirmed with real-time PCR for neurogenic lineage markers. Viable MPIO labeled AFS cells were injected into an acellular nerve allograft (ANA) used to repair a 1.5 cm sciatic nerve defect in 10 rats. Labeled AFS cells were evaluated by MRI at 1, 2, and 4 weeks post-surgery. Intensity of the MPIO regions was quantified using ImageJ. Contiguous frozen sections were stained for iron to identify the labeled AFS cells incorporated into the nerve graft. Co-localization of transplanted cells was confirmed using human specific nuclear antibody (Anti-NuMA).

Results: Labeled AFS cells were viable in vitro(Figure 1). Proliferation rate and morphology between the control and labeled cells demonstrated no significant differences (p=0.58). Cells differentiated towards Schwann-like cells after being cultured in neurogenic induction media. NGF and NEFL gene expression were elevated by fold change of 202.601.89 and 30.621.99, respectively (p<0.005) compared to control. Cytokine quantification analysis of AFS cells showed significantly increased BDNF, ?-NGF, ?-FGF, GDNF, NGF R, NT-4 and TGF-? production. (Fold change compared to undifferentiated control: 10.251.96, 383.0612.93, 3.951.06, 5.781.33, 46.843.67, 2.690.77, 25.393.74, p<0.001 respectively). 7T MRI demonstrated MPIO labeling with a strong decrease in signal, appearing as fuzzy dark spots in T2-weighted images at 4 weeks post-surgery. There was no significant difference in average normalized hypointense region volume between 2 and 4 weeks post-injury (0.470.06 and 0.52 0.12, respectively, Figure 2). Cell integration was confirmed by iron and Anti-NuMA staining.

Conclusions: AFS cells remained viable after labeling and can be used to augment nerve repair by seeding onto ANAs. Cytokine analysis suggests a paracrine-mediated effect on nerve repair. MRI can effectively track the AFS cells longitudinally in the rat model, demonstrating the potential to monitor AFS cell delivery strategies for nerve regeneration.

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