Stroke, Parkinson’s, Alzheimer’s, and other neurological diseases that are relatively frequent in human involve loss of neurons. The advent of tissue regeneration using stem cells holds great promise in finding cures. In particular, mesenchymal stem cells (MSC) appear to be a very potent source for tissue regeneration. Among MSC subtypes, adipose-derived stem cells (ASC) have several distinct advantages. The ASC are abundant, are easy to isolate and expand in vitro, can be used for heterologous as well autologous transplants, and have multilineage differentiation capacity. In addition to osteocytes, chondrocytes, and adipocytes, the ASC have been successfully differentiated into neuronal-like cells by addition of specific neurogenic factors. However, in vivo differentiation of ASC into neurons remains to be demonstrated. In the present study, we used an in vitro system in order to evaluate whether ASC can be induced towards neurogenic lineages by physical contact with freshly isolated neurons or by factors released by neurons without addition of specific neurogenic factors. Experimentally, ASC and neurons (NEU) were extracted from the back fat or the brain, respectively, of a boar transgenic for green fluorescent protein (GFP) or from wild type pigs. The non-GFP neurons were isolated from the brain of 32-day fetuses or adult pigs. Cells were cultivated in 24-well plates with the following combinations: only ASC or NEU in DMEM (controls), ASC with conditioned medium from NEU, or ASC+NEU. Cells were harvested at 24 h and at 3, 7, 14, and 21 days and fixed with 4% paraformaldehyde in PBS for 15 min for immunohistochemistry analysis. After fixation, neuronal differentiation was evaluated by histological staining with specific neuronal markers. The proportion of ASC that differentiated into neuronal-like cells was determined using fluorescence microscopy. We observed little proliferation of ASC in conditioned medium compared with control ASC; however, a few cells exhibited neuronal-like morphology but with no expression of neuronal markers. When ASC were co-cultured with fetal NEU, starting at 3 days, we observed, using microscope analyses, that 4 to 12% of the ASC had neuronal-like morphology and expressed neuron-associated cell markers. When ASC were co-cultured with neurons from adult brain, we observed a lower fraction (between 1 and 2%) of neuronal differentiated cells starting at 7 days. Our data are preliminary but provide evidence that when ASC are in physical contact with neurons (i.e. by cell-to-cell interactions), they can be induced to differentiate into neuronal-like cells. Further, the differentiation is more rapid and extensive when the ASC are in direct contact with fetal neurons. However, further study is necessary to determine whether these neuronal-like cells are functional neurons. In this regard, we are performing electrophysiological analysis and measurement of expression of neuronal genes. In addition, flow cytometry will be used to quantify the proportion of differentiated ASC.
Dual-Channel Fluorescence Imaging of Hydrogel Degradation and Tissue Regeneration in the Brain