Phenotypic and functional characteristics of mesenchymal stem cells differentiated along a Schwann cell lineage

Abstract

We have investigated the phenotypic and bioassay characteristics of bone marrow mesenchymal stromal cells (MSCs) differentiated along a Schwann cell lineage using glial growth factor. Expression of the Schwann cell markers S100, P75, and GFAP was determined by immunocytochemical staining and Western blotting. The levels of the stem cell markers Stro-1 and alkaline phosphatase and the neural progenitor marker nestin were also examined throughout the differentiation process. The phenotypic properties of cells differentiated at different passages were also compared. In addition to a phenotypic characterization, the functional ability of differentiated MSCs has been investigated employing a co-culture bioassay with dissociated primary sensory neurons. Following differentiation, MSCs underwent morphological changes similar to those of cultured Schwann cells and stained positively for all three Schwann cell markers. Quantitative Western blot analysis showed that the levels of S100 and P75 protein were significantly elevated upon differentiation. Differentiated MSCs were also found to enhance neurite outgrowth in co-culture with sensory neurons to a level equivalent or superior to that produced by Schwann cells. These findings support the assertion that MSCs can be differentiated into cells that are Schwann cell-like in terms of both phenotype and function.

Morphological, molecular and functional differences of adult bone marrow- and adipose-derived stem cells isolated from rats of different ages

“…SC, MSC and ASC were harvested from Wistar rats of different ages -new born (neonatal puppies), adult young rat (10 months) and old rats (20 months) -using protocols described elsewhere [2,3]. The animals were killed by cervical dislocation and all procedures were carried out according to the Home Office Act 1986.…”

“…MSC and ASC were stimulated towards glial differentiation using a well established protocol [2,3]. Briefly, subconfluent undifferentiated MSC (uMSC) and ASC (uASC) were cultured in stem cell growth medium (SCGM) containing 1 MM B-mercap-toethanol for 24 h. The cells were washed and the medium replaced with fresh medium supplemented with all-trans-reti-noic acid (35 ng/ml) for 3 days.…”

“…In previous studies, we have shown that adult mesenchymal stem cells originating from either bone marrow (MSC) [2] or from adipose tissue (ASC) [3] can be differ-entiated to SC-like cells. Both differentiated MSC (dMSC) and ASC (dASC) show morphological, biochemical and functional character-istics of SCs, and importantly they secrete growth factors [4,5] and synthesise myelin proteins [6].…”

Neuregulin1 administration increases axonal elongation in dissociated primary sensory neuron cultures

“…DRG neurons were har-vested from adult male Sprague-Dawley rats using a previously described protocol [33]. Dissociated neurons were resuspended in modified Bottenstein and Sato's medium (BSM; F12 medium containing 100 μM putrescine, 30 nM sodium selenite, 20 nM progesterone, 1 mg/ml BSA, 0.1 mg/ml transferrin, 0.01 mM cytosine arabinoside and 10 pM insulin (all Sigma)).…”

“…In the sampling fields, neurite outgrowth was assessed in all cells that were anti-βIII tubulin positive. The following four separate parameters were analysed: number of processes grown from each neuron, length of longest neurite, total neurite area and the percentage of process-bearing neurons (neurite sprouting) [33]. All measurements were carried out using ImageJ tools.…”