A novel combination of factors, termed SPIE, which promotes dopaminergic neuron differentiation from human embryonic stem cells.
BACKGROUND
Stromal-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).


RESULTS
In 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
The 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.
Vazin
Tandis
T
Cellular Neurobiology Research Branch, Intramural Research Program (IRP), National Institute on Drug Abuse (NIDA), NIH, DHHS, Baltimore, MD, USA. vazint@mail.nih.gov
Becker
Kevin G
KG
Chen
Jia
J
Spivak
Charles E
CE
Lupica
Carl R
CR
Zhang
Yongqing
Y
Worden
Lila
L
Freed
William J
WJ
D000818:Animals; D015153:Blotting, Western; D002352:Carrier Proteins / Q000502:physiology*; D002454:Cell Differentiation / Q000502:physiology*; D054377:Chemokine CXCL12 / Q000502:physiology*; D018920:Coculture Techniques; D016207:Cytokines / Q000502:physiology*; D004298:Dopamine / Q000502:physiology*; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D036387:Ephrin-B1 / Q000502:physiology*; D006801:Humans; D007335:Insulin-Like Growth Factor II / Q000502:physiology*; D051379:Mice; D009474:Neurons / Q000166:cytology*; D020411:Oligonucleotide Array Sequence Analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction
D002352:Carrier Proteins; D054377:Chemokine CXCL12; D016207:Cytokines; D036387:Ephrin-B1; C066845:pleiotrophin; D007335:Insulin-Like Growth Factor II; D004298:Dopamine