All-trans-retinoid acid induces the differentiation of encapsulated mouse embryonic stem cells into GABAergic neurons

Abstract

Embryonic stem (ES) cells are pluripotent cells that can differentiate into all three main germ layers: endoderm, mesoderm, and ectoderm. Although a number of methods have been developed to differentiate ES cells into neuronal phenotypes such as sensory and motor neurons, the efficient generation of GABAergic interneurons from ES cells still presents an ongoing challenge. Because the main output of inhibitory GABAergic interneurons is the gamma-aminobutyric-acid (GABA), a neurotransmitter whose controlled homeostasis is required for normal brain function, the efficient generation in culture of functional interneurons may have future implications on the treatment of neurological disorders such as epilepsy, autism, and schizophrenia. The goal of this work was to examine the generation of GABAergic neurons from mouse ES cells by comparing an embryoid body-based methodology versus a hydrogel-based encapsulation protocol that involves the use of all-trans-retinoid acid (RA). We observed that 1) there was a 2-fold increase in neuronal differentiation in encapsulated versus non-encapsulated cells and 2) there was an increase in the specificity for interneuronal differentiation in encapsulated cells, as assessed by mRNA expression and electrophysiology approaches. Furthermore, our results indicate that most of the neurons obtained from encapsulated mouse ES cells are GABA-positive (~87%). Thus, these results suggest that combining encapsulation of ES cells and RA treatment provide a more efficient and scalable differentiation strategy for the generation in culture of functional GABAergic interneurons. This technology may have implications for future cell replacement therapies and the treatment of CNS disorders.

Comparative neuronal differentiation of self-renewing neural progenitor cell lines obtained from human induced pluripotent stem cells

“…It will be interesting to study if different type of primary neuron culture should be used to generate specific neurons subtype. The capability of our hNPs to differentiate in different subtypes of neurons is confirmed by the ability of retinoic acid to induce the maturation of inhibitory neurons (Addae et al, 2012). The differentiation potential of these cells into glial cells has been also determined (data not shown).…”

“…Thus, in order to improve the differentiation of GABAergic neurons, we plated EGFP-hNPs onto rat cortical neurons as previously described and treated the coculture with 1μ M retinoic acid starting 24 h after plating (Addae et al, 2012). After 60 days of differentiation, we stained the hNP-derived mature neuronal cells using an anti-GAD67 antibody to detect GABAergic neurons and observed a MAP2, GAD67, and VGAT positive population of cells (Figures 6A,B).…”

Optogenetic control of iPS cell‐derived neurons in 2D and 3D culture systems using channelrhodopsin‐2 expression driven by the synapsin‐1 and calcium‐calmodulin kinase II promoters

“…Encapsulated cells are buffered in vivo from external factors such as antibodies from the host (Serra et al, ). Furthermore, it has been shown that the alginate encapsulation promotes cell growth, differentiation, maturation, and protein secretion of various cell types including mesenchymal stem cells, mouse ESCs, and human ESCs (Addae et al, ; Serra et al, ). Despite success in the growth support of many cell types, studies utilising alginate encapsulation for hiPSC differentiation and 3D culture are limited (Addae et al, ; Chayosumrit et al, ).…”

RARβ2-dependent signaling represses neuronal differentiation in mouse ES cells

“…Undifferentiated ES cells were maintained on gelatin-coated dishes in ES medium (Dulbecco’s Modified Eagle medium supplemented with 10% ES qualified fetal calf serum (Atlanta Biologicals) as previously described (Addae et al, 2012). In addition, 1 × 10 3 U/ml leukemia inhibitory factor (LIF, Esgro, Millipore) was added to the medium.…”