Nitric oxide signaling and neural stem cell differentiation in peripheral nerve regeneration

Abstract

Objective: The objective was to examine whether nitric oxide signaling plays a role in human embryonic stem cell differentiation into neural cells. This article reviews current literature on nitric oxide signaling and neural stem cell differentiation for potential therapeutic application to peripheral nerve regeneration.

Methods: Human embryonic H9-stem cells were grown, maintained on mitomycin C-treated mouse embryonic fibroblast feeder layer, cultured on Matrigel to be feeder-free, and used for all the experiments. Fluorescent dual-immunolabeling and confocal image analysis were used to detect the presence of the neural precursor cell markers nestin and nitric oxide synthase-1. Fluorescence-activated cell sorting analysis was used to determine the percentage of expression.

Results: We have shown the confocal image of stage 1 human embryonic stem cells coexpressing nestin and nitric oxide synthase-1. Fluorescence-activated cell sorting analysis indicated 24.3% positive labeling of nitric oxide synthase-1. Adding retinoic acid (10(-6) M) to the culture medium increased the percent of nitric oxide synthase-1 positive cells to 33.9%. Combining retinoic acid (10(-6) M) with 8-brom cyclic guanosine monophosphate (10(-5) M), the fluorescence-activated cell sorting analysis demonstrated a further increase of nitric oxide synthase-1 positive cells to 45.4%. Our current results demonstrate a prodifferentiation potency of nitric oxide synthase-1, stimulated by retinoic acid with and without cyclic guanosine monophosphate.

Conclusion: We demonstrated for the first time how nitric oxide/cyclic guanosine monophosphate signaling contributes to the development of neural precursors derived from human embryonic stem cells and enhances the differentiation of precursors toward functional neurons for peripheral nerve regeneration.

Figure 1

Nitric oxide signaling pathway. NO indicates nitric oxide; sGC, soluble guanylate cyclase; GTP, Guanine triphosphate; cGMP, cyclic Guanine monophosphate; PDE, phosphodiesterase; PKG, protein kinase G.

Figure 2

Immunofluorescence labeling of human embryonic stem cell (hESC)–derived neural stem cells (NSCs) showing coexpression of neural stem cell marker, nestin, and nitric oxide synthase (NOS)-1 in stage 1 hESC. Nestin-PE (red) and NOS-1 FITC (green) expression in stage 1 hESC-5x (A). Nestin and NOS-1 expression in hESC derived NSCs in differentiation medium-20x (B). Nestin and NOS-1 expression in hESCs treated with retinoic acid (10⁻⁶ M) (C). Combined treatment of retinoic acid (10⁻⁶ M) and 8-brom-cGMP (10⁻⁵ M) (D). Fluorescence activated cell sorter analysis of stage 1–differentiated hESCs for CD-133 and NOS-1 (E-F). Right panel (E) showing 97.5% of cells coexpressing CD-133 and NOS-1 (darker color). Left panel (F) was the result of negative control.