A method to generate human mesenchymal stem cell-derived neurons which express and are excited by multiple neurotransmitters

Abstract

The present study describes a protocol to generate heterogenous populations of neurotransmitter-producing neurons from human mesenchymal stem cells (MSCs). MSCs are bone marrow (BM)-derived cells which undergo lineage- specific differentiation to generate bone, fat, cartilage and muscle, but are also capable of transdifferentiating into defined ectodermal and endodermal tissues. The purpose of this study is to evaluate the potential of MSCs as an alternative source of customized neurons for experimental neurobiology or other regenerative approaches. Our neuronal protocol utilizes freshly harvested human MSCs cultured on specific surfaces and exposed to an induction cocktail consisting of low serum concentration, retinoic acid (RA), growth factors and supplements. Here we report on the types of neurotransmitters produced by the neurons, and demonstrate that the cells are electrically responsive to exogenous neurotransmitter administration.

Keywords: Gamma-aminobutyric acid; Mesenchymal stem cells.

Fig. 1. Neuropeptide expression in uninduced and induced MSCs.

A. Uninduced (D0) and induced (D12) MSCs were co-labeled with PE-anti-SP and FITC-anti-SV2 or FITC-anti-synaptophysin (Syn), and then counterlabeled with nucleus-specific DAPI. Figure shows representative labelings of five different experiments. Images are shown at 10X magnification. B. Media from uninduced (D0) and induced (D6 and D12) MSCs was immunoprecipitated with anti-VIP and anti-CGRP and then analyzed by western blot. Representative blots are shown for three different experiments. Band densities were quantified and normalized to total protein. Results are presented as mean ± SD relative fold change, where the lowest value is arbitrarily assigned a value of 1. C. Uninduced and induced MSCs were labeled at 2 day intervals, up to 12 days, with PE-anti-CGRP and then counterlabeled with DAPI. Relative fluorescence intensities were normalized to values for uninduced cells, which were arbitrarily assigned a value of 1. Figure shows representative labelings of five different experiments. Images are shown at 10X magnification. D. Whole cell extracts from D0, D6 and D12 cells were prepared and analyzed by western blots with anti-Leu-Enkephalin. Normalizations were performed with anti-β-actin. Representative blots are shown for three different experiments. Band quantification was performed as in B *p<0.05 vs. D12 induced cells **p<0.05 vs. uninduced cells

Fig. 2. Expression of CNS neurotransmitters in uninduced and induced MSCs.

A. Whole cell extracts from uninduced (D0) and induced (D6 and D12) MSCs were prepared and analyzed by western blots with anti-tyrosine hydroxylase (TyrH), -tryptophan hydroxylase (TrypH), -glutamic acid decarboxylase (GAD) and -vesicular acetylcholine transporter (VAChT). Normalizations were performed with anti-β-actin. Human brain extract served as positive control. Representative blots are shown for three different experiments. Band densities were quantified and normalized to total protein. Results are presented as mean ± SD relative fold change, where the lowest value is arbitrarily assigned a value of 1. B. D12 induced cells were labeled with PE-anti-GAD and then counter-labeled with DAPI. Figure shows representative labelings of five different experiments. Images are shown at 20X magnification. C. Uninduced and induced MSCs were labeled at 2 day intervals, up to 12 days, with PE-anti-glutamate and then counterlabeled with DAPI. Relative fluorescence intensities were normalized to values for uninduced cells, which were arbitrarily assigned a value of 1. Figure shows representative labelings of five different experiments. Images are shown at 10X magnification.*p<0.05 vs. uninduced cells **p<0.05 vs. D12 induced cells

Fig. 3. Colocalization of SP and GAD in induced MSCs.

D12 cells were co-labeled with FITC-anti-GAD and PE-anti-SP, and then counter-labeled with DAPI. Figure shows representative labelings of five different experiments. Images are shown at 40X magnification

Fig. 4. Expression of GABA and glutamate receptors in uninduced (D0) and induced (D12) MSCs. A.

Total RNA from D0 and D12 cells was studied for expression of the GABAA A receptor β1 subunit and NMDA1 receptor by RT-PCR. Normalizations were performed with oligonucleotides specific for GAPDH. Representative gel is shown for three different experiments. B. Whole cell extracts from D0 and D12 cells were prepared and analyzed by western blots with anti-GABAAR-β1 and anti-NMDAR1. Normalizations were performed with anti-β-actin. Representative blots are shown for three different experiments.

Fig. 5. GABA and glutamate elicit responses in D12 induced MSCs.

A. FM1-43 FX lipophilic plasma membrane dye was added at 5 µg/mL to D12 cells. Cells were cultured on glass coverslips, with dye incubation lasting for 1 min. Cells were fixed with ice-cold 3.7% formaldehyde and then examined for plasma membrane staining. Intracellular vesicle dye reuptake was studied by treating cells with 1 mM GABA (left panel) or 1 mM glutamate (middle panel) following labeling. Vehicle controls used 1% BSA (right panel). Figure represents four different experiments, each performed with cells from a different donor. Representative inward currents elicited by 1 mM GABA (B) or 1 mM glutamate (C) from cells in culture for 12 days. The peak amplitude is 49 pA for GABA-induced current, and 81 pA for glutamate-induced current. Whole-cell currents were recorded at a holding potential of -50 mV