Heterogeneous population of dopaminergic neurons derived from mouse embryonic stem cells: preliminary phenotyping based on receptor expression and function

Abstract

The possibility exists that directed differentiation of mouse embryonic stem (mES) cells is capable of yielding enriched populations of dopaminergic neurons, but at present there is little understanding of the pharmacological properties of these cells; or whether such cells represent a pharmacologically, phenotypically similar population. In this study we used a simple culture protocol to generate dopaminergic neurons and offer a preliminary pharmacological investigation of these cells using Ca2+ imaging and [3H]-dopamine release studies. In fluo-4 AM loaded cells, 13-17 days postplating, and after the addition of tetrodotoxin some of the population of mouse embryonic stem cell-derived neurons responded to adenosine triphosphate (ATP), noradrenaline (NA), acetylcholine (ACh) and L-glutamate (L-glut) with elevations of Ca2+ influx. Within the microtubule-associated protein and tyrosine hydroxylase (TH)-positive cell population adenosine triphosphate, noradrenaline, acetylcholine and L-glutamate elicited positive elevations of Ca2+ in 74, 66, 58 and 67% of the population; cells could be further subdivided into three major pharmacologically distinct populations based on the combinations of agonist they responded to. Acetylcholine (30 microM) and noradrenaline (30 microM) were the only agonists to elicit significant tritium overflow from [3H]-dopamine loaded cells. The acetylcholine effect was blocked by atropine (1 microM) and tetrodotoxin (1 microM) and elevated by haloperidol (100 nM). The noradrenaline effects were reduced by cocaine (10 microM), but not by tetrodotoxin (100 nM). These data indicate that the dopaminergic neurons derived from mouse embryonic stem cells represent a heterogeneous population possessing combinations of purinergic, adrenergic, cholinergic and glutamatergic receptors located on the cell soma.

In Vitro Maturation of Dopaminergic Neurons Derived from Mouse Embryonic Stem Cells: Implications for Transplantation

“…Figure 1A shows a bright field image of cells at day 21, with TH + cells overlaid in red and cells loaded with fluo-4 in blue. Consistent with a previous study from this laboratory [22], fluo-4 AM loaded mouse ESC-derived TH + neurons responded to ATP (300 µM), NA (30 µM), ACh (30 µM), Glut (30 µM) and KCl (30 mM) with elevations of [Ca 2+ ] i . Figure 1B shows typical TH + neuron responses to ATP (300 µM) at days 13 and 23 of differentiation.…”

“…Background light was subtracted from each region and emission intensity calculated using Metafluor (Universal Imaging, USA). After a 10-min equilibration one of the agonists, either adenosine triphosphate (ATP, 300 µM), noradrenaline (NA, 30 µM), acetylcholine (ACh, 30 µM), L-glutamate (Glut, 30 µM), or γ-aminobutyric acid (GABA, 30 µM) was added to the culture well and the cells left for 60 s. Agonist concentrations were based on previous work from this laboratory [20], [21], [22], [23]. Cells were then washed with fresh pre-warmed buffer thrice.…”

“…In previous studies from this laboratory we have shown that a variety of neurotransmitter agonists elevated intracellular Ca 2+ ([Ca 2+ ] i ) in tyrosine hydroxylase positive (TH + ) derived from mouse embryonic stem cells [20], [21], [22]. As these cultures develop there is an increase in post-synaptic inhibitory (presumably GABAergic) currents [20], probably due to the GABAergic neuronal population that develops alongside the TH + population [21], [23].…”

Drug discovery in Parkinson's disease: update and developments in the use of cellular models

“…DA neurons have been differentiated from mouse ESCs by several protocols, including co-culturing with stromal PA-6 cells[61, 62] and embryoid body-based lineage selection[63]. DA neurons derived from mouse ESCs have pharmacological phenotypes similar to primary DA neurons[64]. Furthermore, transplantation of ES-derived neural precursors or DA neurons rescues behavioral deficits in animal PD models[65–67].…”

The role of glial cells in influencing neurite extension by dorsal root ganglion cells

“…Cells cultured on glass coverslips were preserved in kyrofix (ethanol:H 2 O:PEG 300 at 7.9:7.4:1) at 48C until immunocytochemistry was performed (Raye et al, 2007). Following washing with PBS, then fixing in ice-cold acetone-methanol (1:1) for 30 min, cells were then permeabilized with 0.03% Triton X-100 and blocked with 3% donkey serum (Jackson ImmunoResearch Laboratories, West Grove, PA, USA).…”