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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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Morphological comparisons between non-induced MSC, chemically-induced MSC, chemically-induced NIH 3T3 and primary neurons.A) Schematic illustration of the chemical neuronal induction protocol. B–D) Phase-contrast microscopy images showing morphological aspects. B) MSC have a spread-out cytoplasm in standard medium; C) after induction for 8 h cells exhibited neuronal-like morphology; D) fibroblast NIH 3T3 after 8 h induction adopted neuronal-like morphology; E) primary neuronal cultures. Scale bar = 100 µm.
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pone-0005222-g001: Morphological comparisons between non-induced MSC, chemically-induced MSC, chemically-induced NIH 3T3 and primary neurons.A) Schematic illustration of the chemical neuronal induction protocol. B–D) Phase-contrast microscopy images showing morphological aspects. B) MSC have a spread-out cytoplasm in standard medium; C) after induction for 8 h cells exhibited neuronal-like morphology; D) fibroblast NIH 3T3 after 8 h induction adopted neuronal-like morphology; E) primary neuronal cultures. Scale bar = 100 µm.

Mentions: Adult rat bone marrow-derived MSC were isolated and successfully expanded, displaying characteristic morphology and colony formation [34]. Morphological alterations were observed a few hours after MSC induction following the protocol proposed by Woodbury et al. [9] (figure 1), thus reproducing the effects described previously. Those cells showed morphological similarities to neurons. Changes in cell shape were gradual and due to retraction of cytoplasmic processes. A long period of induction (24 h) caused complete processes retraction and cells acquired a round shape, leading to detachment from the culture plate surface. To evaluate cell death we performed Annexin V and PI staining, as demonstrated below. To properly evaluate cell death and detachment, experiments were done at 8 h post-induction, when 80% of the initial amount of cells was likely to be alive and 82% displayed a neuronal-like shape (figure 1).


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)

Morphological comparisons between non-induced MSC, chemically-induced MSC, chemically-induced NIH 3T3 and primary neurons.A) Schematic illustration of the chemical neuronal induction protocol. B–D) Phase-contrast microscopy images showing morphological aspects. B) MSC have a spread-out cytoplasm in standard medium; C) after induction for 8 h cells exhibited neuronal-like morphology; D) fibroblast NIH 3T3 after 8 h induction adopted neuronal-like morphology; E) primary neuronal cultures. Scale bar = 100 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0005222-g001: Morphological comparisons between non-induced MSC, chemically-induced MSC, chemically-induced NIH 3T3 and primary neurons.A) Schematic illustration of the chemical neuronal induction protocol. B–D) Phase-contrast microscopy images showing morphological aspects. B) MSC have a spread-out cytoplasm in standard medium; C) after induction for 8 h cells exhibited neuronal-like morphology; D) fibroblast NIH 3T3 after 8 h induction adopted neuronal-like morphology; E) primary neuronal cultures. Scale bar = 100 µm.
Mentions: Adult rat bone marrow-derived MSC were isolated and successfully expanded, displaying characteristic morphology and colony formation [34]. Morphological alterations were observed a few hours after MSC induction following the protocol proposed by Woodbury et al. [9] (figure 1), thus reproducing the effects described previously. Those cells showed morphological similarities to neurons. Changes in cell shape were gradual and due to retraction of cytoplasmic processes. A long period of induction (24 h) caused complete processes retraction and cells acquired a round shape, leading to detachment from the culture plate surface. To evaluate cell death we performed Annexin V and PI staining, as demonstrated below. To properly evaluate cell death and detachment, experiments were done at 8 h post-induction, when 80% of the initial amount of cells was likely to be alive and 82% displayed a neuronal-like shape (figure 1).

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