Over the past decade, stem cells from adult bone marrow have been exploited as therapeutic vectors in the treatment of a wide variety of diseases [4
]. This study clearly shows the plasticity of BMSCs by their differentiation into Schwann-like cells with the typical spindle-shaped Schwann cell morphology. In this study we have confirmed that the BMSCs express characteristic mesenchymal stem cell (MSC) surface markers (CD44 and CD90 positive, but CD31 and CD45 negative) and also demonstrated the multilineage potential of BMSCs [13
]. Clear evidence is provided to show that rat BMSCs have the ability to differentiate along a glial lineage and express cell markers (S100, P75 and GFAP) which are typical for glial cells including Schwann cells [3
On the basis of our results, the sequential administration of various factors, BME, RA, followed by a mixture of FSK, bFGF, PDGF and HRG, effectively induces the differentiation of BMSCs into Schwann-like cells. The phenotype of the differentiated BMSCs resembled that of Schwann cells. The previous reports showed that the addition of BME to cultured BMSCs induced the formation of neurite-like processes [14
]. RA is a morphogenic factor, and was reported to induce differentiation of neural stem cells into nerve cells [15
]. Therefore, BME and RA are presumed to work as triggering factors, inducing changes in the morphological and transcriptional characteristics of BMSCs.
Previous studies reported that bFGF functions as a mitogen for BMSCs, and that MAP kinases are activated by PDGF and bFGF in MSCs [16
]. HRG, a subtype of neuregulin, instructively influences the decision of cell fates and was reported to induce neural crest cells to develop selectively into Schwann cells [17
FSK increases the level of intracellular cyclic adenosine monophosphate (cAMP), which increases mitogenic responses at an early time [18
]. As cAMP elevation is reported to enhance the responsiveness of cells to trophic factors [19
], FSK together with bFGF, PDGF, and HRG could have a synergistic effect in enhancement of these factors to BMSCs [20
MTT assay and flow cytometry analysis for annexin V and PI indicated that during the chemical induction process there was an increase in cell death. Putative early apoptotic cells increased after exposure to BME and RA.
Measures of cell death during this specific induction process are indicative of toxicity of the chemical compounds used. Here we show that almost 50% of cells die within 48 h of chemical induction, in agreement with other studies [8
Our results indicate that RA exerts two apparently opposite effects on cultured ADSCs, i.e., a differentiating effect and induction of cell death. Flow cytometry analysis for annexin V and PI indicated that putative early apoptotic cells increased after exposure to RA. These data agreed with previous reports where all-trans-retinoic acid produced similar effects in cancers [22
] and stroke [23
The mechanism of the apoptotic effects of β-mercaptoethanol and all-trans-retinoic acid is unknown and there are several pathways that need to be investigated.
Oliva et al.
reported that antiproliferative activity of retinoic acid is associated with (and probably due to) the up-regulation of two pivotal cdk inhibitors, and subsequent cdk2 activity decrease and retinoblastoma protein (pRb) hypophosphorylation [24
pRb is a tumour suppressor protein that is dysfunctional in many types of cancer [25
]. In the hypophosphorylated state, pRb is active and carries out its role as a tumour suppressor by inhibiting cell cycle progression [26
However, there is evidence indicating that either direct contact with axons or survival factors secreted by neurons are necessary for the survival and development of SC precursors as well as of mature Schwann cell [8
In conclusion, these findings indicated that BMSCs could differentiate into Schwann-like cells in terms of morphology and phenotype. As a side effect of differentiation an increased cell death rate was noted and our findings indicate that the principle mode of cell death is by apoptosis.
Hence, a considerable amount of BMSCs would die during differentiation into Schwann-like cells, depending on the method used. This may compromise the optimal outcome of the engraftment and subsequently nerve regeneration and repair.