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.
Highlights
There is a great interest in the possibility of using human embryonic stem cells to produce specific cell types which might be used either in cellular therapy or as in vitro models of human cells
In order to test the abilities of mouse embryonic fibroblasts (MEF), MM55K, MS5, PA6-X1, PA6-X and PA6-DA cell lines to induce DA induction of human ESC (hESC), small colonies of BG01V2 were co-cultured with each of the feeder cell lines for 12 days
Neurons expressing b-III-tubulin were detected in 565% and 363% of colonies grown on MEF and MM55K respectively, whereas co-culture with PA6-DA cells resulted in 82613% b-III-tubulin+ colonies
Summary
There is a great interest in the possibility of using human embryonic stem cells (hESC) to produce specific cell types which might be used either in cellular therapy or as in vitro models of human cells. The most commonly-used technique for producing DA neurons from ESC requires a co-culture step, most often using stromal cells such as the mouse PA6 cell line, but in some cases human astrocytes or other cell lines [8,9,10,11,12,13,14,15,16,17,18]. A second method involves the formation of embryoid bodies (EBs), in which case internal factors, produced by hESC, are presumably responsible for the early induction phase. This approach involves a complex series of procedures including enzymatic digestion and various isolation techniques followed by SHH and FGF8 exposure [2,5]. We found that factors secreted by PA6 cells provided lineage-specific instructions to induce DA differentiation of human ESC (hESC)
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