In Vivo Tracking of Amniotic Fluid Derived Stem Cells on Acellular Nerve Graft

Abstract

Purpose

This study aimed to track amniotic fluid derived stem (AFS) cells that are seeded into nerve allografts, and to elucidate the potential mechanisms of their impact on the regenerating nerve.

Methods

Amniotic fluid derived stem cells were labeled using supra-paramagnetic micrometer-sized iron oxide (MPIO) and assessed for viability. Cells were cultured in neurogenic induction media and evaluated for neurogenic growth factors. Differentiated cells were confirmed with reverse transcription-polymerase chain reaction for neurogenic lineage markers. MPIO labeled AFS cells were injected into an acellular nerve allograft (ANA) to repair sciatic nerve defects in rats. Labeled AFS cells were evaluated using magnetic resonance imaging (MRI) and the intensity of the MPIO regions in the ANA was quantified. Contiguous frozen sections were stained for iron to identify cells incorporated into the nerve graft.

Results

Proliferation rate and morphology between the control and MPIO-labeled cells demonstrated similar patterns. AFS cells differentiated toward Schwann-like cells after being cultured in neurogenic induction media. nerve growth factor (NGF) and neurofilament light chain gene expression were elevated by 202.60 ± 1.89 and 30.62 ± 1.99 times, respectively, compared with control. Cytokine quantification analysis showed significantly increased brain-derived neurotrophic factor, β-NGF, β-fibroblast growth factor, glial cell-derived neurotrophic factor, nerve growth factor receptor, neurotrophin-4, and tumor growth factor β production. 7-Tesla MRI demonstrated MPIO labeling with a strong decrease in the T2-weighted signal. The average normalized hypointense region volume was similar between groups postinjury. Cell integration into ANA was confirmed by iron staining.

Conclusions

AFS cells remained viable after labeling with MPIO and can be used to augment nerve repair by seeding onto ANAs. Cytokine analysis suggests a paracrine-mediated effect of AFS cells on nerve repair following injury. MRI can effectively track the AFS cells longitudinally in the rat model up to 4 weeks postimplantation.

Clinical relevance

The study provides translational evidence on using MRI for tracking AFS cells in the decellularized nerve allografts for nerve repair and regeneration.