Limits...
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.

Show MeSH

Related in: MedlinePlus

Electrophysiological recordings of Na+ current and AP firing ability.A) Representative voltage-dependent Na+ current recorded during a 100-ms hyperpolarizing pulse, followed by 200-ms depolarizing steps (10-mV increments) (see inset) for primary culture neuron (A1), and chemically-induced MSC (A2). B) Membrane potential of chemically-induced MSC recorded under current clamp. Note the inability to fire AP with depolarizing current steps (50 or 1000 ms).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2667250&req=5

pone-0005222-g008: Electrophysiological recordings of Na+ current and AP firing ability.A) Representative voltage-dependent Na+ current recorded during a 100-ms hyperpolarizing pulse, followed by 200-ms depolarizing steps (10-mV increments) (see inset) for primary culture neuron (A1), and chemically-induced MSC (A2). B) Membrane potential of chemically-induced MSC recorded under current clamp. Note the inability to fire AP with depolarizing current steps (50 or 1000 ms).

Mentions: To determine whether neuronal-like cells derived from adult rat MSC were electrically active, we performed patch-clamp recordings to study the presence of K+ and Na+ currents (whole-cell voltage-clamp), voltage-gated channels (cell attached) and the ability of cells to fire action potentials (whole-cell current-clamp). We observed that both neurons from E17 cortex primary culture (n = 6) and untreated MSC (n = 5) exhibited K+ currents (Figue 6 A1, A2, B1, B2). However no K+ current was detected from chemically-induced MSC with neuronal-like morphology (n = 7) (figure 7 A3, A4, B1, B2). These cells also did not present Na+ current when compared with controls (neurons from primary culture n = 6; chemically-induced MSC n = 10) (figure 8 A1, A2). We observed that both, the immature neurons from primary culture and the chemically-induced MSC were unable to fire action potentials when depolarizing currents were injected in current-clamp recordings (n = 3; n = 5) (figure 8 B).


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)

Electrophysiological recordings of Na+ current and AP firing ability.A) Representative voltage-dependent Na+ current recorded during a 100-ms hyperpolarizing pulse, followed by 200-ms depolarizing steps (10-mV increments) (see inset) for primary culture neuron (A1), and chemically-induced MSC (A2). B) Membrane potential of chemically-induced MSC recorded under current clamp. Note the inability to fire AP with depolarizing current steps (50 or 1000 ms).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0005222-g008: Electrophysiological recordings of Na+ current and AP firing ability.A) Representative voltage-dependent Na+ current recorded during a 100-ms hyperpolarizing pulse, followed by 200-ms depolarizing steps (10-mV increments) (see inset) for primary culture neuron (A1), and chemically-induced MSC (A2). B) Membrane potential of chemically-induced MSC recorded under current clamp. Note the inability to fire AP with depolarizing current steps (50 or 1000 ms).
Mentions: To determine whether neuronal-like cells derived from adult rat MSC were electrically active, we performed patch-clamp recordings to study the presence of K+ and Na+ currents (whole-cell voltage-clamp), voltage-gated channels (cell attached) and the ability of cells to fire action potentials (whole-cell current-clamp). We observed that both neurons from E17 cortex primary culture (n = 6) and untreated MSC (n = 5) exhibited K+ currents (Figue 6 A1, A2, B1, B2). However no K+ current was detected from chemically-induced MSC with neuronal-like morphology (n = 7) (figure 7 A3, A4, B1, B2). These cells also did not present Na+ current when compared with controls (neurons from primary culture n = 6; chemically-induced MSC n = 10) (figure 8 A1, A2). We observed that both, the immature neurons from primary culture and the chemically-induced MSC were unable to fire action potentials when depolarizing currents were injected in current-clamp recordings (n = 3; n = 5) (figure 8 B).

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