A Novel Combination of Factors, Termed SPIE, which Promotes Dopaminergic Neuron Differentiation from Human Embryonic Stem Cells

Abstract

BackgroundStromal-Derived Inducing Activity (SDIA) is one of the most efficient methods of generating dopaminergic (DA) neurons from embryonic stem cells (ESC). DA neuron induction can be achieved by co-culturing ESC with the mouse stromal cell lines PA6 or MS5. The molecular nature of this effect, which has been termed “SDIA” is so far unknown. Recently, we found that factors secreted by PA6 cells provided lineage-specific instructions to induce DA differentiation of human ESC (hESC).Methodology/Principal FindingsIn the present study, we compared PA6 cells to various cell lines lacking the SDIA effect, and employed genome expression analysis to identify differentially-expressed signaling molecules. Among the factors highly expressed by PA6 cells, and known to be associated with CNS development, were stromal cell-derived factor 1 (SDF-1/CXCL12), pleiotrophin (PTN), insulin-like growth factor 2 (IGF2), and ephrin B1 (EFNB1). When these four factors, the combination of which was termed SPIE, were applied to hESC, they induced differentiation to TH-positive neurons in vitro. RT-PCR and western blot analysis confirmed the expression of midbrain specific markers, including engrailed 1, Nurr1, Pitx3, and dopamine transporter (DAT) in cultures influenced by these four molecules. Electrophysiological recordings showed that treatment of hESC with SPIE induced differentiation of neurons that were capable of generating action potentials and forming functional synaptic connections.Conclusions/SignificanceThe combination of SDF-1, PTN, IGF2, and EFNB1 mimics the DA phenotype-inducing property of SDIA and was sufficient to promote differentiation of hESC to functional midbrain DA neurons. These findings provide a method for differentiating hESC to form DA neurons, without a requirement for the use of animal-derived cell lines or products.

A simple method for large‐scale generation of dopamine neurons from human embryonic stem cells

“…Several prior studies have analyzed factors secreted from PA6 stromal cells and have suggested those that might be effective at inducing or maintaining dopamine neurons from ESCs. Vazin et al (2009) performed genome expression analysis by comparing PA6 cells with other cell lines and concluded that four factors were mainly responsible for PA6 cell-derived effects on ESCs: stromal cell-derived factor 1 (SDF-1/CXCL12), pleiotrophin (PTN), insulin-like growth factor 2 (IGF2), and ephrin B1 (EFNB1). They used a combination of these four factors to induce differentiation of TH-positive neurons successfully from human ESCs (Vazin et al, 2009).…”

“…Vazin et al (2009) performed genome expression analysis by comparing PA6 cells with other cell lines and concluded that four factors were mainly responsible for PA6 cell-derived effects on ESCs: stromal cell-derived factor 1 (SDF-1/CXCL12), pleiotrophin (PTN), insulin-like growth factor 2 (IGF2), and ephrin B1 (EFNB1). They used a combination of these four factors to induce differentiation of TH-positive neurons successfully from human ESCs (Vazin et al, 2009). Wnt5a is also important for the development of DA neurons (Parish et al, 2008) and is expressed by PA6 cells (Hayashi et al, 2008).…”

Attractive and repulsive factors act through multi-subunit receptor complexes to regulate nerve fiber growth

“…The multivalent binding of a ligand (simultaneous binding to one assembly of receptors) has recently been shown to drive receptor clustering at the nanoscale level (Conway et al, 2013). Super-resolution microscopy has been used to visualize ephrin-B2-driven clustering of the EphB4 receptor at the nanoscale level on adult neuronal stem cells, as ephrin-B-EphB signaling has been shown to regulate neuronal stem cell differentiation (Ashton et al, 2012;Conway et al, 2013;Vazin et al, 2009). The multivalency of a recombinant ephrin-B2 ectodomain conjugated to a soluble biopolymer strongly enhanced the bioactivity of ligands, resulting in the induction of neuronal stem cell signaling -i.e.…”

Dopaminergic Neurons Derived from Human Embryonic Stem Cell Derived Neural Progenitors: Biological Relevance and Application

“…An Lmx1a promoter was used in hESCs to promote differentiation of 10 to 20% TH+ neurons (Friling et al, 2009). Efforts to improve the derivation of dopaminergic neurons have included formation of spherical neural masses (SNMs) instead of EBs prior to differentiation (Cho et al, 2008, Vazin et al, 2009. Elucidating the factors expressed in and secreted by stromal cells used to differentiate dopaminergic neurons included microarray studies.…”

“…Additionally, receptors for these genes were more highly expressed in NSCs compared to hESCs. A study using various 398 combinations of factors which activated these receptors or replicated the factors expressed by PA6 cells determined that the combination of factors termed SPIE (SDF-1, PTN, IGF2, and EFNB1) was most effective at differentiating hESCs towards dopaminergic neurons (Cho et al, 2008, Vazin et al, 2009). However, a highly efficient and effective method of dopaminergic differentiation has not been obtained.…”