Study of lead-induced neurotoxicity in cholinergic cells differentiated from bone marrow-derived mesenchymal stem cells.

The developing brain is susceptible to the neurotoxic effects of lead. Exposure to lead has main effects on the cholinergic system and causes reduction of cholinergic neuron function during brain development. Disruption of the cholinergic system by chemicals, which play important roles during brain development, causes of neurodevelopmental toxicity. Differentiation of stem cells to neural cells is recently considered a promising tool for neurodevelopmental toxicity studies. This study evaluated the toxicity of lead acetate exposure during the differentiation of bone marrow-derived mesenchyme stem cells (bone marrow stem cells, BMSCs) to CCholinergic neurons. Following institutional animal care review board approval, BMSCs were obtained from adult rats. The differentiating protocol included two stages that were pre-induction with ß-mercaptoethanol (BME) for 24 h and differentiation to cholinergic neurons with nerve growth factor (NGF) over 5 days. The cells were exposed to different lead acetate concentrations (0.1-100 µm) during three stages, including undifferentiated, pre-induction, and neuronal differentiation stages; cell viability was measured by MTT assay. Lead exposure (0.01-100 µg/ml) had no cytotoxic effect on BMSCs but could significantly reduce cell viability at 50 and 100 µm concentrations during pre-induction and neuronal differentiation stages. MAP2 and choline acetyltransferase (ChAT) protein expression were investigated by immunocytochemistry. Although cells treated with 100 µm lead concentration expressed MAP2 protein in the differentiation stages, they had no neuronal cell morphology. The ChAT expression was negative in cells treated with lead. The present study showed that differentiated neuronal BMSCs are sensitive to lead toxicity during differentiation, and it is suggested that these cells be used to study neurodevelopmental toxicity.

Study of the chlorpyrifos neurotoxicity using neural differentiation of adipose tissue-derived stem cells.

Chlorpyrifos (CPF) is the most commonly used organophosphorus insecticide which causes neurodevelopmental toxicity. So far, animals have been used as ideal models for neurotoxicity studies, but working with animals is very expensive, laborious, and ethically challenging. This has encouraged researchers to seek alternatives. During recent years, several studies have reported successful differentiation of embryonic and adult stem cells to neurons. This has provided an excellent model for neurotoxicologic studies. In this study, neural differentiation of mouse adipose tissue-derived stem cells (ADSCs) was used as an in vitro model for investigation of CPF neurotoxicity. For this purpose, mouse ADSCs were cultured in a medium containing knockout serum replacement and were treated with different concentrations of CPF at several stages of differentiation. Cytotoxic effect of CPF and the expression of neuron-specific genes and proteins were studied in the differentiating ADSCs. Furthermore, the activity of acetylcholinesterase was assessed by Ellman assay at different stages of differentiation. This study showed that up to 500 µM CPF did not alter viability of the undifferentiated ADSCs, whereas viability of the differentiating cells decreased with 500 µM CPF. CPF upregulated the expression of some neuron-specific genes and seemed to decrease the number of ß-tubulin III and MAP2 proteins-expressing cells. There was no detectable acetylcholine esterase activity in differentiated ADSCs. In summary, it was shown that CPF treatment can decrease the viability of ADSC-derived neurons and dysregulate the expression of some neuronal markers through acetylcholinesterase-independent mechanisms. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1510-1519, 2016.

Study of lead-induced neurotoxicity in neural cells differentiated from adipose tissue-derived stem cells.

In recent years, the use of stem cells as a new tool to create an in vitro model for toxicological studies has been considered. Adipose tissue-derived stem cells (ADSCs) are mesenchymal stem cells which have been extracted from adipose tissue by a less invasive method and rapidly propagated in culture medium compared with other sources. These cells have the capacity to differentiate into different cell lineage in vitro including neural cells. The aim of this study was to investigate the effect of lead exposure at various stages of differentiation on the neural differentiation of ADSCs. Third-passaged ADSCs were differentiated to neural cell in differentiation medium during 16 d. The ADSCs were exposed to lead (0.1-100 µg/ml) before differentiation and during differentiation on days 1, 7 and 14. The cell viability was assessed by MTT assay after 48 h. Also expression of ß-tubulin III protein and Nestin, NeuN, NF70, Synaptophysin genes were evaluated at the end of differentiation in all treated groups. The results showed that lead had no effect on viability of undifferentiated ADSCs but differentiating cells showed various sensitivities to lead exposure and cells were more vulnerable to lead exposure at early stage of differentiation. Also, lead exposure at different stages of differentiation had various effects on gene expressions. Our study indicated that neural cells differentiated from ADSCs in vitro are sensitive to neurotoxic effect of lead as well-known developmental neurotoxicant, and then ADSCs could be a candidate as an alternative method for assessing neurodevelopmental toxicity potential of chemicals.