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Chemically-induced RAT mesenchymal stem cells adopt molecular properties of neuronal-like cells but do not have basic neuronal functional properties.

Barnabé GF, Schwindt TT, Calcagnotto ME, Motta FL, Martinez G, de Oliveira AC, Keim LM, D'Almeida V, Mendez-Otero R, Mello LE - PLoS ONE (2009)

Bottom Line: Moreover, increased intracellular cysteine after treatment indicates an impairment of redox circuitry during chemical induction, and in vitro electrophysiological recordings (patch-clamp) of the chemically induced MSC did not indicate neuronal properties as these cells do not exhibit Na(+) or K(+) currents and do not fire action potentials.Our findings suggest that a disruption of redox circuitry plays an important role in this specific chemical induction protocol, which might result in cytoskeletal alterations and loss of functional ion-gated channels followed by cell death.Despite the neuronal-like morphology and neural protein expression, induced rat bone marrow MSC do not have basic functional neuronal properties, although it is still plausible that other methods of induction and/or sources of MSC can achieve a successful neuronal differentiation in vitro.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Fisiologia, Universidade Federal de São Paulo (UNIFESP), São Paulo, São Paulo, Brazil.

ABSTRACT
Induction of adult rat bone marrow mesenchymal stem cells (MSC) by means of chemical compounds (beta-mercaptoethanol, dimethyl sulfoxide and butylated hydroxyanizole) has been proposed to lead to neuronal transdifferentiation, and this protocol has been broadly used by several laboratories worldwide. Only a few hours of MSC chemical induction using this protocol is sufficient for the acquisition of neuronal-like morphology and neuronal protein expression. However, given that cell death is abundant, we hypothesize that, rather than true neuronal differentiation, this particular protocol leads to cellular toxic effects. We confirm that the induced cells with neuronal-like morphology positively stained for NF-200, S100, beta-tubulin III, NSE and MAP-2 proteins. However, the morphological and molecular changes after chemical induction are also associated with an increase in the apoptosis of over 50% of the plated cells after 24 h. Moreover, increased intracellular cysteine after treatment indicates an impairment of redox circuitry during chemical induction, and in vitro electrophysiological recordings (patch-clamp) of the chemically induced MSC did not indicate neuronal properties as these cells do not exhibit Na(+) or K(+) currents and do not fire action potentials. Our findings suggest that a disruption of redox circuitry plays an important role in this specific chemical induction protocol, which might result in cytoskeletal alterations and loss of functional ion-gated channels followed by cell death. Despite the neuronal-like morphology and neural protein expression, induced rat bone marrow MSC do not have basic functional neuronal properties, although it is still plausible that other methods of induction and/or sources of MSC can achieve a successful neuronal differentiation in vitro.

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Related in: MedlinePlus

Analysis of the expression of NeuN, NF-200 and β-tubulin III by Western Blot. Lanes: 1) non-induced NIH 3T3; 2) chemically-induced 3T3; 3) non-induced MSC; 4) serum-free MSC; 5 and 6) chemically-induced MSC for 8 h; 7, 8 and 9) freshly extracted bone marrow mononuclear cells; 10) brain tissue (positive control).Actin (43 KDa) was used as an internal control.
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pone-0005222-g004: Analysis of the expression of NeuN, NF-200 and β-tubulin III by Western Blot. Lanes: 1) non-induced NIH 3T3; 2) chemically-induced 3T3; 3) non-induced MSC; 4) serum-free MSC; 5 and 6) chemically-induced MSC for 8 h; 7, 8 and 9) freshly extracted bone marrow mononuclear cells; 10) brain tissue (positive control).Actin (43 KDa) was used as an internal control.

Mentions: Considering the Western Blot analysis (figure 4), samples from non-induced NIH 3T3 (lane 1) and non-induced MSC (lane 3) expressed only the 66 KDa NeuN isoform, while bone marrow mononuclear cells (lanes 7, 8, 9) and brain (lane 10) extracts had all three NeuN isoforms (66, 46, 48 KDa). The antibody against β-tubulin III cross reacts with the rat tubulin, and explained the 55 KDa band. Only bone marrow mononuclear cells (lanes 7, 8, 9) and brain (lane 10) samples had the 48 KDa band corresponding to the β-tubulin III expression. NF-200 expression increased after MSC chemical induction (lanes 5, 6) and is also present in bone marrow mononuclear cells (lanes 7, 8, 9). A small increase in NF-200 expression was observed after serum deprivation of NIH 3T3 cells (lane 2) and of MSC (lane 4). Actin (43 KDa) was used as an internal control.


Chemically-induced RAT mesenchymal stem cells adopt molecular properties of neuronal-like cells but do not have basic neuronal functional properties.

Barnabé GF, Schwindt TT, Calcagnotto ME, Motta FL, Martinez G, de Oliveira AC, Keim LM, D'Almeida V, Mendez-Otero R, Mello LE - PLoS ONE (2009)

Analysis of the expression of NeuN, NF-200 and β-tubulin III by Western Blot. Lanes: 1) non-induced NIH 3T3; 2) chemically-induced 3T3; 3) non-induced MSC; 4) serum-free MSC; 5 and 6) chemically-induced MSC for 8 h; 7, 8 and 9) freshly extracted bone marrow mononuclear cells; 10) brain tissue (positive control).Actin (43 KDa) was used as an internal control.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2667250&req=5

pone-0005222-g004: Analysis of the expression of NeuN, NF-200 and β-tubulin III by Western Blot. Lanes: 1) non-induced NIH 3T3; 2) chemically-induced 3T3; 3) non-induced MSC; 4) serum-free MSC; 5 and 6) chemically-induced MSC for 8 h; 7, 8 and 9) freshly extracted bone marrow mononuclear cells; 10) brain tissue (positive control).Actin (43 KDa) was used as an internal control.
Mentions: Considering the Western Blot analysis (figure 4), samples from non-induced NIH 3T3 (lane 1) and non-induced MSC (lane 3) expressed only the 66 KDa NeuN isoform, while bone marrow mononuclear cells (lanes 7, 8, 9) and brain (lane 10) extracts had all three NeuN isoforms (66, 46, 48 KDa). The antibody against β-tubulin III cross reacts with the rat tubulin, and explained the 55 KDa band. Only bone marrow mononuclear cells (lanes 7, 8, 9) and brain (lane 10) samples had the 48 KDa band corresponding to the β-tubulin III expression. NF-200 expression increased after MSC chemical induction (lanes 5, 6) and is also present in bone marrow mononuclear cells (lanes 7, 8, 9). A small increase in NF-200 expression was observed after serum deprivation of NIH 3T3 cells (lane 2) and of MSC (lane 4). Actin (43 KDa) was used as an internal control.

Bottom Line: Moreover, increased intracellular cysteine after treatment indicates an impairment of redox circuitry during chemical induction, and in vitro electrophysiological recordings (patch-clamp) of the chemically induced MSC did not indicate neuronal properties as these cells do not exhibit Na(+) or K(+) currents and do not fire action potentials.Our findings suggest that a disruption of redox circuitry plays an important role in this specific chemical induction protocol, which might result in cytoskeletal alterations and loss of functional ion-gated channels followed by cell death.Despite the neuronal-like morphology and neural protein expression, induced rat bone marrow MSC do not have basic functional neuronal properties, although it is still plausible that other methods of induction and/or sources of MSC can achieve a successful neuronal differentiation in vitro.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Fisiologia, Universidade Federal de São Paulo (UNIFESP), São Paulo, São Paulo, Brazil.

ABSTRACT
Induction of adult rat bone marrow mesenchymal stem cells (MSC) by means of chemical compounds (beta-mercaptoethanol, dimethyl sulfoxide and butylated hydroxyanizole) has been proposed to lead to neuronal transdifferentiation, and this protocol has been broadly used by several laboratories worldwide. Only a few hours of MSC chemical induction using this protocol is sufficient for the acquisition of neuronal-like morphology and neuronal protein expression. However, given that cell death is abundant, we hypothesize that, rather than true neuronal differentiation, this particular protocol leads to cellular toxic effects. We confirm that the induced cells with neuronal-like morphology positively stained for NF-200, S100, beta-tubulin III, NSE and MAP-2 proteins. However, the morphological and molecular changes after chemical induction are also associated with an increase in the apoptosis of over 50% of the plated cells after 24 h. Moreover, increased intracellular cysteine after treatment indicates an impairment of redox circuitry during chemical induction, and in vitro electrophysiological recordings (patch-clamp) of the chemically induced MSC did not indicate neuronal properties as these cells do not exhibit Na(+) or K(+) currents and do not fire action potentials. Our findings suggest that a disruption of redox circuitry plays an important role in this specific chemical induction protocol, which might result in cytoskeletal alterations and loss of functional ion-gated channels followed by cell death. Despite the neuronal-like morphology and neural protein expression, induced rat bone marrow MSC do not have basic functional neuronal properties, although it is still plausible that other methods of induction and/or sources of MSC can achieve a successful neuronal differentiation in vitro.

Show MeSH
Related in: MedlinePlus