Protocol 1 (P#1): Ham’s F12 (Sigma Aldrich) supplemented with 20 ng/mL epidermal growth factor (EGF) (PeproTech) and 20 ng/mL basic fibroblast growth factor (bFGF) (PeproTech, Cranbury, NJ, USA), until the cells detached as neurospheres from the surface. We chose three different protocols for the neural differentiation from the literature, which differ in terms of the duration, surface coating, and media composition. The cells in Passage 5 were used for all the experiments. Upon reaching subconfluency, the cells were detached from the surface using a trypsin/EDTA (ethylenediaminetetraacetic acid) solution (Pan-Biotech). Blood cells and other nonplastic-adherent cells were removed by washing with phosphate-buffered saline (PBS) (Sigma Aldrich). The cell pellet was washed, the cells were seeded into T175-cell-culture flasks (Greiner Bio One, Kremsmünster, Austria), and they were allowed to adhere in Eagle’s minimum essential medium (MEM) (Sigma Aldrich), supplemented with 20% fetal bovine serum (Pan-Biotech, Aidenbach, Germany) and a penicillin–streptomycin solution (Sigma Aldrich) (referred to as the proliferation medium below). Afterwards, the cell suspension was passed through a 100 µm filter (MERCK Millipore, Billerica, MA, USA) and was centrifuged for 5 min at 500 g. Louis, MO, USA) per mL of fat tissue at 37° C for 60 min. Briefly, freshly harvested adipose tissues from the inguinal fat pads of rats were minced into small pieces (1 mm 3), and were subsequently digested with 2U collagenase (Sigma Aldrich, St. ĪSCs were isolated as previously described. However, they can also be bioengineered to release growth factors, or could be used as a scaffold for the introduction of regenerative cells. In their simplest implementation, these conduits are simply bridging the gap in the critical-size nerve defects, giving the axon the right direction, and protecting the regeneration site from interfering substances. These artificial tubes are used for guiding axonal regrowth, and they can be manufactured from a variety of materials, ranging from simple proteins, such as extracellular matrix components (collagen, fibronectin, laminin), to complex materials, such as spider silk. In order to transfer these data, the results were reproduced on surfaces that could be used as nerve guidance conduits. Three distinct differentiation protocols were investigated for their ability to induce the expressions of specific glial cell markers, as well as the secretion of neurotropic and neurotrophic factors, both at the transcriptional and translational levels. In this study, various culture conditions were investigated for their ability to efficiently direct ASCs into specialized SC phenotypes. Nevertheless, therapeutic applications should consider these diverse phenotypes as a potential approach for stem-cell-based nerve-injury treatment.Īlthough various studies have applied SC-like ASCs for the purpose of peripheral nerve regeneration, precise phenotypes of SC-like ASCs have found little attention yet. It remains uncertain what features of these SC-like cells contribute the most to adequate functional recovery during the different phases of nerve recovery. The NGF secretion affected the neurite outgrowth significantly. These results were reproducible when the ASCs were differentiated on surfaces potentially used for nerve guidance conduits. One protocol yielded relatively high expression rates of neurotrophins, whereas another protocol induced myelin-marker expression. The dASCs were highly diverse in their expression profiles. Additionally, the influence of the medium conditioned by dASCs on a neuron-like cell line was evaluated.
The differentiated ASCs (dASCs) were compared for their expressions of neurotrophins (NGF, GDNF, BDNF), myelin markers (MBP, P0), as well as glial-marker proteins (S100, GFAP) by RT-PCR, ELISA, and Western blot. In this study, three differentiation protocols were investigated for their ability to differentiate ASCs from rats into specialized SC phenotypes. Lately, adipose-tissue-derived stem cells (ASCs) differentiated towards SC (Schwann cell)-like cells seem to fulfill some of the needs for ameliorated nerve recovery. The lack of supportive Schwann cells in segmental nerve lesions seems to be one cornerstone for the problem of insufficient nerve regeneration.
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