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

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.

Figure 1. 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.

Figure 2. This panel shows the pattern of neural markers expression in the non-induced, chemically-induced and serum-free MSC groups.

Primary neuronal cultures were used as positive controls for the immunocytochemistry. Phase microscopy images of cells stained for: β-tubulin III (β-tubIII), MAP-2, NeuN, NF-200, NSE, S100 and GFAP. Scale bar = 100 µm.

Figure 3. Proportion of positive stained cells for neural proteins in rat bone marrow mononuclear cells and in non-induced, serum-free and chemically-induced MSC.

For each neural marker, statistically significant differences (ANOVA, p<0.05) between cell types/conditions appears under brackets.

Figure 4. 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.

Figure 5. 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.

Figure 6. Ratio between Hcy e Cys contents obtained for non-induced (control, CTR) and chemically-induced MSC.

No cellular change in Hcy content was observed in the induction protocol (p = 0.23). Total Cys content increased almost four times (p = 0.00006) in chemically-induced MSC compared to non-induced ones. Data represented as mean±standard deviation.

Figure 7. Electrophysiological recordings of K⁺ current.

A) Voltage-dependent K⁺ current recorded from primary culture neuron (A1), non-induced MSC (A2) and chemically-induced MSC (A3 and A4). B) I/V curves calculated from traces in A showing decreased K⁺ current for treated MSC (B1, B2). C) Single-channel K⁺ current recordings showing frequent channel opening in primary culture neurons (C1) but absent in chemically-induced MSC (C2).

Figure 8. 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).