Elsevier

Cytotherapy

Volume 9, Issue 2, 2007, Pages 170-183
Cytotherapy

Generating neuron-like cells from BM-derived mesenchymal stromal cells in vitro

https://doi.org/10.1080/14653240701196829Get rights and content

Background

The multipotency of stromal cells has been studied extensively. It has been reported that mesenchymal stromal cells (MSC) are capable of differentiating into cells of multilineage. Different methods and reagents have been used to induce the differentiation of MSC. We investigated the efficacy of different growth factors in inducing MSC differentiation into neurons.

Methods

MSC from human BM were isolated and cultured in media supplemented with 10% FBS. These cells were identified and later induced to differentiate into neuron-like cells using different neurotrophic factors. Three different growth factors were used, either alone or in combination: brain-derived neurotrophic factor, epidermal growth factor and neural growth factor.

Results

After 10 days of culture, MSC showed neuron-like morphologic changes. Immunostaining showed that these cells expressed markers for neurons (growth-associated protein-43, neuron-specific nuclear protein and neurofilament 200 kDa) and expression of these markers suggested the transition of immature stages to more mature stages of neuron-like cells.

Discussion

Our results show that BM-derived MSC can differentiate not only into target cells of mesodermal origin but also neuron-like cells of ectodermal origin. The findings show that a combination of growth factors is more effective in inducing MSC into neuron-like cells.

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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