Generating neuron-like cells from BM-derived mesenchymal stromal cells in vitro
Introduction
Stromal cells are thought to be the major source of growth factors for development of hematopoietic stem cells, and interact closely with hematopoietic stem cells within the BM microenvironment [1]. Lately, these stromal cells, and a subset also known as mesenchymal stem cells, have been studied extensively for their ability to differentiate into target cells. It has been reported that mesenchymal stromal cells (MSC) are capable of differentiating into adipocytes, osteocytes and chondrocytes 2., 3., 4., 5., 6., 7., 8., 9.. MSC have also been shown to differentiate into myocardiocytes 10., 11., 12. and neuron-like cells [13, 14].
Different methods have been used to coax stromal cells to differentiate into neuron-like cells, such as using chemical reagents β-mercaptoethanol (BME), DMSO and butylated hydroxyanisole (BHA), growth factors and 5-Aza-C [15]. In this study, we investigated the efficacy of different neutrotrophic factors in generating neuron-like cells from BM-derived MSC, alone and in combination.
Section snippets
Sampling
BM aspirate, obtained from patients with informed consent, was used in this study. The samples were obtained in accordance with the protocol approved by the Ethics and Research Committee of The National University of Malaysia.
BM preparation and cell culture
BM aspirate was diluted with equal amounts of PBS prior to centrifugation with Ficoll-Pague (Amersham Biosciences, Uppsala, Sweden). The mononuclear cells were washed and suspended in DMEM-LG (Sigma-Aldrich, St Louis, MO, USA) before the viability of the cells was
Isolation and expansion of MSC
Adherent cells grown on culture flasks displayed typical morphologies of undifferentiated MSC. Figure 1 shows that long spindle-shaped adherent cells colonized the whole surface, while some flatten morphology was also observed. Further chemical staining showed that the adherent cells stained positively towards NSE and PAS (Figure 2). The results obtained were similar to the characteristics of MSC.
Immunophenotyping on MSC
Flow cytometry was used to determine the phenotype of MSC. Cell-surface markers of MSC from passage
Discussion
MSC were characterized by their ability to proliferate in culture, with a tendency to adhere to the plastic culture flask, and by the presence of a consistent set of protein markers or surface Ag determinants. The results in this study show that MSC can be readily isolated from mononuclear cells of BM aspirate. The initial culture of the mononuclear cells generated a layer of heterogeneous, adherent cells, with at least two different morphologies. This process of generating adherent cells took
Acknowledgements
We acknowledge the generous support from the Ministry of Science, Technology and Innovation (MOSTI), Malaysia, through the IRPA mechanism and MAKNA (National Cancer Council) of Malaysia. We thank the staff and fellow researchers from the Stem Cell Unit, National University of Malaysia, for their encouragement and assistance.
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