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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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Cell death analysis of MSC.Graphic representations of the percentage of MSC that are: A) live cells (Annexin− PI−); B) necrotic (Annexin− PI+); C) early apoptotic (Annexin+ PI−); or D) late apoptotic (Annexin+ PI+) in the non-induced (control, CTR), serum-free (SF), pre-induced (BME) or chemically-induced (for CI4 h, CI8 h and CI24 h). Mean±standard error. p<0.05, # different from every other group; * different from non-induced and serum-free.
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pone-0005222-g005: Cell death analysis of MSC.Graphic representations of the percentage of MSC that are: A) live cells (Annexin− PI−); B) necrotic (Annexin− PI+); C) early apoptotic (Annexin+ PI−); or D) late apoptotic (Annexin+ PI+) in the non-induced (control, CTR), serum-free (SF), pre-induced (BME) or chemically-induced (for CI4 h, CI8 h and CI24 h). Mean±standard error. p<0.05, # different from every other group; * different from non-induced and serum-free.

Mentions: Flow cytometry analysis for Annexin V and PI indicated that during the chemical induction process there was an increase in cell death (figure 5). Although serum deprivation for 24 h is characterized as a stressful condition for MSC, it was not sufficient to trigger cell death mechanisms. Furthermore, serum-free MSC show the same proportions of live and dead cells as non-induced MSC. At the end of the chemical induction protocol (24 h) we found a significant percentage of dead cells (44.8%; figure 4A; ANOVA p<0.05, followed by Newman-Keuls as compared to every other group, p<0.01). Our results also demonstrated that most of cells died due to apoptotic events during chemical induction. Indeed, Annexin V−/PI+ cells, likely to indicate necrotic elements, were less frequent than Annexin V+ (both PI+ and PI−) likely to indicate apoptotic events. Putative early apoptotic cells (Annexin V+/PI−) increased after exposure to BME and BHA/DMSO at 4 h, and were augmented after exposure to BHA/DMSO at later times with the highest value occurring at 24 h (ANOVA p<0.05, followed by Newman-Keuls all groups as compared to non-induced group, and chemically-induced for 24h as compared to every other group, p<0.01; figure 4C). The number of late apoptotic cells (figure 4D) significantly increased after BME pre-induction treatment (11%) as compared to non-induced MSC and at the 24 h induction time point (24.2%) as compared to every other group. We have thus found a number of significant differences between non-induced and chemically-induced MSC groups for live and apoptotic cells (early or late apoptotic; figure 5).


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)

Cell death analysis of MSC.Graphic representations of the percentage of MSC that are: A) live cells (Annexin− PI−); B) necrotic (Annexin− PI+); C) early apoptotic (Annexin+ PI−); or D) late apoptotic (Annexin+ PI+) in the non-induced (control, CTR), serum-free (SF), pre-induced (BME) or chemically-induced (for CI4 h, CI8 h and CI24 h). Mean±standard error. p<0.05, # different from every other group; * different from non-induced and serum-free.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0005222-g005: Cell death analysis of MSC.Graphic representations of the percentage of MSC that are: A) live cells (Annexin− PI−); B) necrotic (Annexin− PI+); C) early apoptotic (Annexin+ PI−); or D) late apoptotic (Annexin+ PI+) in the non-induced (control, CTR), serum-free (SF), pre-induced (BME) or chemically-induced (for CI4 h, CI8 h and CI24 h). Mean±standard error. p<0.05, # different from every other group; * different from non-induced and serum-free.
Mentions: Flow cytometry analysis for Annexin V and PI indicated that during the chemical induction process there was an increase in cell death (figure 5). Although serum deprivation for 24 h is characterized as a stressful condition for MSC, it was not sufficient to trigger cell death mechanisms. Furthermore, serum-free MSC show the same proportions of live and dead cells as non-induced MSC. At the end of the chemical induction protocol (24 h) we found a significant percentage of dead cells (44.8%; figure 4A; ANOVA p<0.05, followed by Newman-Keuls as compared to every other group, p<0.01). Our results also demonstrated that most of cells died due to apoptotic events during chemical induction. Indeed, Annexin V−/PI+ cells, likely to indicate necrotic elements, were less frequent than Annexin V+ (both PI+ and PI−) likely to indicate apoptotic events. Putative early apoptotic cells (Annexin V+/PI−) increased after exposure to BME and BHA/DMSO at 4 h, and were augmented after exposure to BHA/DMSO at later times with the highest value occurring at 24 h (ANOVA p<0.05, followed by Newman-Keuls all groups as compared to non-induced group, and chemically-induced for 24h as compared to every other group, p<0.01; figure 4C). The number of late apoptotic cells (figure 4D) significantly increased after BME pre-induction treatment (11%) as compared to non-induced MSC and at the 24 h induction time point (24.2%) as compared to every other group. We have thus found a number of significant differences between non-induced and chemically-induced MSC groups for live and apoptotic cells (early or late apoptotic; figure 5).

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