Original research articleDifferentiation of adult rat mesenchymal stem cells to GABAergic, dopaminergic and cholinergic neurons
Introduction
The central nervous system is well known for its limited ability to regenerate after an injury. Cell replacement therapy should be investigated to develop more effective and long-term therapies.
Of the adult stem cells, mesenchymal stem cells (MSCs) appear to be the best candidates for regenerative medicine. MSCs are easily isolated from bone marrow. A previous study showed that only 0.001–0.01% of mononuclear cells are MSCs [1] but this type of cell can easily be increased in vitro. MSCs are multipotent cells of a mesenchymal origin that can differentiate into cells from all three germ layers in the appropriate environmental conditions. In addition, a pervious study found that undifferentiated MSCs express genes that are typical for ectodermal, endodermal and mesodermal cells [2], thus suggesting the high plasticity of MSCs. MSCs are able to be converted into a clonogenic neural stem cell-like population that grow in neurosphere-like structures. This step is probably crucial in MSCs differentiation into cells with the morphological and functional characteristics of neural, astroglial and oligodendroglial cells [3]. MSCs seem to be ideal candidates for cell therapy across an allogenic barrier because of their immune suppressive functions [4]. Although, autologous transplantation would be ideal, allogenic ones are more probable in neurodegenerative disorders because the number of MSCs decreases with age [5] and their ability to differentiate also decreases. A small number of MSCs express the CXCR4 (chemokine receptor type 4) receptor and as a result can migrate into SDF-1 (stromal derived factor-1) niches. MSCs with a migration capability seem to be the best population to investigate because after transplantation they can migrate to precisely the damaged region [6].
Several studies have demonstrated the ability of MSCs to trans-differentiate into different kinds of neurons but the culture conditions that are required to create a homogenous population of a specific type of neurons are still unknown. The presence of the Cxcr4 receptor on adult neurons has not yet been investigated as well. The aim of the current work was to differentiate rat MSCs (rMSCs) into GABAergic, cholinergic and dopaminergic cells. It is well known that the differentiation effect depends on the environmental conditions and this is why various differentiation cocktails will be used to culture a homogenous population of one phenotype of neurons. Co-expression neuronal markers and the Cxcr4 receptor was investigated as well.
Section snippets
Culture of rats MSCs
rMSCs were isolated from the femurs and tibias of adult Wistar rats as was previously described [7] and then maintained in a Dulbecco's modified Eagle's medium – nutrient mixture F-12 (DMEM/F12; PAA) supplemented with 10% fetal bovine serum (FBS; PAA) and a 1% Antibiotic Antimycotic Solution (100X) (Sigma–Aldrich). Non-adherent cells were removed for the first time after 24 h and adherent ones were cultured in 10 ml fresh cultured medium. Non-adherent cells were centrifuged at 400 × g for 10 min at 4
Isolation and culturing of rMSC
Rats MSCs were isolated using a combined method – Histopaque® separation and plastic adherence. The cell suspensions were layered on Histopaque® and subsequently seeded in a tissue culture flask. After two days, many of the rounded as well as spindle-shaped cells had attached to the base of the tissue culture flask (Fig. 3A). The majority of the adherent cells displayed a spindle-like shape (Fig. 3B). These cells began to proliferate and form small colonies. The cells continued to grow, the
Discussion
The first extremely important stage of our experiment was the neuronal induction, which should have directed MSCs to the differentiation of neuronal cells. Two mitogens, rhEGF and rhbFGF, were used in this protocol. A previous study investigated 30 ng/ml EGF induced MSCs differentiation to neuronal cells in in vitro conditions [8] and the infusion of EGF and bFGF into the brains of mice in order to stimulate neurogenesis in the hippocampal dentate subgranular zone and the subventricular zone [9]
Conflicts of interest
There are no conflicts of interest.
Funding
This work was supported by a grant from Medical University of Silesia, Poland (KNW-1-002/D/2/0 to Paulina Borkowska).
References (26)
- et al.
A Simple and efficient method for deriving neurospheres from bone marrow stromal cells
Biochem Biophys Res Commun
(2008) - et al.
Bone marrow stromal cells as replacement cells for Parkinson's disease: generation of an anatomical but not functional neuronal phenotype
Transl Res
(2011) - et al.
Adult rat mesenchymal stem cells differentiate into neuronal-like phenotype and express a variety of neuro-regulatory molecules in vitro
Neurosci Res
(2010) - et al.
Adult human bone marrow stromal spheres express neuronal traits in vitro and in a rat model of Parkinson's disease
Brain Res
(2006) - et al.
Two isoforms of glutamate decarboxylase: why?
Trends Pharmacol Sci
(1998) - et al.
Glutamic acid decarboxylase 65 i 67 expression in the lateral septum is up-regulated in association with the postpartum period in mice
Brain Res
(2012) - et al.
Transdifferentiation of bone marrow stromal cells into cholinergic neuronal phenotype: a potential source for cell therapy in spinal cord injury
Cytotherapy
(2009) - et al.
Differential regulation of the high affinity choline transporter and the cholinergic locus by cAMP signaling pathways
Brain Res
(2007) - et al.
Multilineage potential of adult human mesenchymal stem cells
Science
(1999) - et al.
Adult bone marrow stromal stem cells express germline, ectodermal, endodermal and mesodermal genes prior to neurogenesis
J Neurosci Res
(2002)
Efficient generation of neural stem cell-like cells from adult human bone marrow stromal cells
J Cell Sci
Vetro-like activity of mesenchymal stem cells: functional discrimination between cellular responses to alloantigens and recall antigens
J Immunol
Bone marrow as a source of circulating CXCR4+ tissue-committed stem cells
Biol Cell
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