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Adipose-derived stem cells (AScs) can easily be obtained and expanded in vitro for use in autologous cell therapy. Via their production of cytokines and neurotrophic factors, transplanted AScs provide neuroprotection, neovascularization and induction of axonal sprouting. However, the influencing mechanism of undifferentiated AScs on nerve regeneration is currently only partially understood. In the present study, undifferentiated AScs and cutaneous primary afferent dorsal root ganglion (dRG) neurons were co-cultured in order to investigate their interaction. AScs were isolated from adult rat fat tissue. The presence of characteristic stem cell markers was determined by flow cytometry in three subsequent passages. Adipogenic, osteogenic, chondrogenic and glial differentiation was performed in order to evaluate their differentiation capacity. A direct co-culture system with dRG cells was established to determine the effect of undifferentiated pluripotent AScs on neurite elongation. Neurite outgrowth, length and number was examined in the co-culture and compared with single-culture cells and cells stimulated with nerve growth factor (NGF). In ASc cultures, NGF expression was assessed by ELISA. The present results demonstrated that the specific mesenchymal stem cell surface markers cd44, cd73 and cd90 were detected in all three subsequent passages of the isolated ASCs. In accordance, ASc differentiation into adipogenic, osteogenic, chondrogenic and Schwann cell phenotype was conducted successfully. Neurite outgrowth of dRG neurons was enhanced following co-culture with AScs, resulting in increased neurite length after 24 h of cultivation. Furthermore, neurite outgrowth of dRG neurons was directed towards the undifferentiated ASc and direct cell-to-cell contact was observed. In summary, the results of the present study revealed an interaction between the two cell types with guidance of neurite growth towards the undifferentiated ASC. These findings suggest that the use of undifferentiated ASc optimizing tissue-engineered constructs may be promising for peripheral nerve repair.

IntroductionAdipose-derived stroma cells (ASCs) are attractive cells for cell-based gene therapy but are generally difficult to transfect. Nucleofection has proven to be an efficient method for transfection of primary cells. Therefore, we used this technique to transfect ASCs with a vector encoding for Ambystoma mexicanum epidermal lipoxygenase (AmbLOXe) which is a promising bioactive enzyme in regenerative processes. Thereby, we thought to even further increase the large regenerative potential of the ASCs.MethodsASCs were isolated from the inguinal fat pad of Lewis rats and were subsequently transfected in passage 1 using Nucleofector® 2b and the hMSC Nucleofector kit. Transfection efficiency was determined measuring co-transfected green fluorescent protein (GFP) in a flow cytometer and gene expression in transfected cells was detected by reverse transcription polymerase chain reaction (RT-PCR). Moreover, cell migration was assessed using a scratch assay and results were tested for statistical significance with ANOVA followed by Bonferroni’s post hoc test.ResultsHigh initial transfection rates were achieved with an average of 79.8 ± 2.82% of GFP positive cells although longer cultivation periods reduced the number of positive cells to below 5% after four passages. Although successful production of AmbLOXe transcript could be proven the gene product had no measureable effect on cell migration.ConclusionsOur study demonstrates the feasibility of ASCs to serve as a vehicle of AmbLOXe transport for gene therapeutic purposes in regenerative medicine. One potential field of applications could be peripheral nerve injuries.

Introduction: Regenerative Peripheral Nerve Interfaces (RPNIs) are neurotized muscle grafts that control prostheses through electromyography (EMG). RPNI signals have not been quantified during phases of voluntary movements. This study: a) characterizes active RPNI signaling compared to background activity and b) defines the reliability and validity of RPNI function during gait phases of rat walking. Material and Methods: Rat groups were: Control (n=3), RPNI (n=3), Denervated (n=3). Bipolar electrodes were implanted onto the soleus muscles in each group. The Control group was left intact. The Denervated group had the tibial nerve transected. For RPNIs, the soleus muscle was freely grafted to the ipsilateral thigh and neurotized by the transected tibial nerve. While walking on a treadmill, rats were videographed and raw EMG signals were simultaneously recorded. Outcome measurements were integrated EMG (iEMG) and iEMG normalized (NiEMG) to stance, swing, or sit gait phase. Results: Majority of EMG activity was observed within the stance phase-70% for Control and 79% for RPNI-as expected for active soleus postural muscles. Stance NiEMG signals were greater than swing NiEMG averages for Control and RPNI groups (Fig 1). The Denervated group stance and swing NiEMG signals were not different without peripheral nerve control. Fidelity of RPNI stance activity (NiEMG signal to background signal) was 5.6 to 1, or double the Control signal fidelity. Correlations between iEMG and stance time for the Control (r=0.74) and RPNI (r=0.76) indicate strong signal reliability (Fig. 2). Conclusion: Measurements of fidelity, reliability, and validity for RPNI signal detection all exceeded normal probability (p<0.05) during voluntary movement. LOP32: Enhancement of neuritic outgrowth in vitro by adipose-derived stromal cells

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