Differentiation of adult rat mesenchymal stem cells to GABAergic, dopaminergic and cholinergic neurons

doi:10.1016/j.pharep.2014.08.022

Pharmacological Reports

Volume 67, Issue 2, April 2015, Pages 179-186

https://doi.org/10.1016/j.pharep.2014.08.022 Get rights and content

Abstract

Background

Mesenchymal stem cells (MSCs) are able to differentiate into cells from all three germ layers. The aim of the current work was the differentiation rat MSCs into GABAergic, cholinergic and dopaminergic cells.

New method

In this paper, we present differentiation cocktails with a hippocampal astrocyte conditioned medium and with a glioblastoma conditioned medium. We wanted to maximize the role of endogenous secreted substances by cells from the central nervous system in both combinations. These modifications create a microenvironment of differentiation that is similar to natural conditions. Moreover, the presence of the Cxcr4 receptor on neuron-like cells was investigated first time.

Results

Our results show that a differentiation cocktail with a hippocampal astrocyte conditioned medium is the most effective and that 17% Gad67(+) and 7% Acht(+) cells were observed using this protocol. After differentiation using the glioblastoma conditioned medium, 12% Gad67 (+) was observed. The presence of the Cxcr4 migration receptor on Gad67(+) and Th(+) cells were observed, which might suggest the transplantation potential of differentiated cells.

Comparison with existing methods

Our results are slightly lower than those of previous studies but when differences in counting cells is taken into account, a comparison of results is really difficult.

Conclusions

These new differentiation cocktails should be further investigated and in the next experiment only a part of MSCs that expressed the Cxcr4 receptor will be differentiated. We suppose that the Cxcr4(+) cells may differentiate more easily and as a result, we may achieve a homogenous population of one phenotype of 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).

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Original research articleDifferentiation of adult rat mesenchymal stem cells to GABAergic, dopaminergic and cholinergic neurons

Abstract

Background

New method

Results

Comparison with existing methods

Conclusions

Introduction

Section snippets

Culture of rats MSCs

Isolation and culturing of rMSC

Discussion

Conflicts of interest

Funding

Biochem Biophys Res Commun

Transl Res

Neurosci Res

Brain Res

Trends Pharmacol Sci

Brain Res

Cytotherapy

Brain Res

Multilineage potential of adult human mesenchymal stem cells

Science

Adult bone marrow stromal stem cells express germline, ectodermal, endodermal and mesodermal genes prior to neurogenesis

J Neurosci Res

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

Original research article
Differentiation of adult rat mesenchymal stem cells to GABAergic, dopaminergic and cholinergic neurons