title: Kinetic analysis of neurotrophin-3-mediated differentiation of embryonic stem cells into neurons.
abstract: The goal of this study was to develop a kinetic analysis that could predict the behavior of embryonic stem cell-derived neural progenitor cells (ESNPCs) in response to treatment with neurotrophin-3 (NT-3). Previous studies have shown that NT-3 activates the mitogen-activated protein (MAP) kinase cascade in embryonic stem cells and promotes differentiation of ESNPCs into neurons. MAP kinase activation after NT-3 stimulation was confirmed experimentally, and a kinetic analysis was developed using rate constants obtained from the literature. Concentrations of select signaling components were estimated for ESNPCs using real-time reverse transcription polymerase chain reaction by comparing mRNA levels to those of cell types with known protein concentrations. This assumption was validated using Western blots, and incorporated into the analysis. This analysis was used to predict the minimum NT-3 concentration necessary to promote neuronal differentiation of ESNPCs based on the activation of MAP kinase. These predictions were then tested experimentally to confirm the validity of the analysis. Finally, expression of the transcription factor mammalian achate schute homolog 1 and beta-tubulin III (an early neuronal marker) was examined in response to the different NT-3 doses to confirm the link between MAP kinase activation and neuronal differentiation. Overall, this study provides insight into the kinetics of the intracellular processes that promote ESNPC differentiation to neurons.
authors.affiliation: Department of Biomedical Engineering, Washington University, St. Louis, Missouri 63130, USA.
authors.forename: Stephanie M
authors.initials: SM
authors.lastname: Willerth
authors.forename: Shelly E
authors.initials: SE
authors.lastname: Sakiyama-Elbert
mesh_terms: D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000378:metabolism; D015153:Blotting, Western; D002454:Cell Differentiation / Q000187:drug effects*; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000187:drug effects* / Q000201:enzymology; D004789:Enzyme Activation / Q000187:drug effects; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D009474:Neurons / Q000166:cytology* / Q000187:drug effects* / Q000201:enzymology; D020933:Neurotrophin 3 / Q000494:pharmacology*; D051381:Rats; D015203:Reproducibility of Results
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D048049:Extracellular Signal-Regulated MAP Kinases; D020933:Neurotrophin 3; C499317:Ascl1 protein, mouse
references.citation: Development. 2006 Mar;133(5):889-900
references.pmid: 16452094
references.citation: Neuroscience. 2008 Jan 2;151(1):138-47
references.pmid: 18093741
references.citation: Neuron. 2005 Jan 20;45(2):189-92
references.pmid: 15664170
references.citation: Bioinformatics. 2005 Mar15;21(6):741-53
references.pmid: 15479714
references.citation: Tissue Eng. 2005 Mar-Apr;11(3-4):506-12
references.pmid: 15869429
references.citation: Exp Cell Res. 2005 Jun 10;306(2):343-8
references.pmid: 15925590
references.citation: Nat Med. 1999 Dec;5(12):1410-2
references.pmid: 10581084
references.citation: Blood. 2000 Aug 15;96(4):1215-22
references.pmid: 10942360
references.citation: FEBS Lett. 2000 Oct 6;482(3):169-74
references.pmid: 11024454
references.citation: Stem Cells. 2002;20(2):119-38
references.pmid: 11897869
references.citation: Nat Biotechnol. 2002 Apr;20(4):370-5
references.pmid: 11923843
references.citation: J Neurosci Res. 2003 Mar 1;71(5):648-58
references.pmid: 12584723
references.citation: J Neurosci. 2003 Jun 15;23(12):5149-60
references.pmid: 12832539
references.citation: J Recept Signal Transduct Res. 2003;23(2-3):197-209
references.pmid: 14626447
references.citation: Biochem Biophys Res Commun. 2004 Feb 20;314(4):1113-20
references.pmid: 14751248
references.citation: Proc Natl Acad Sci U S A. 2004 Mar 2;101(9):2900-5
references.pmid: 14978270
references.citation: Trends Biotechnol. 2004 Apr;22(4):155-6
references.pmid: 15104108
references.citation: Biophys J. 2004 May;86(5):2748-57
references.pmid: 15111393
references.citation: Biotechnol Appl Biochem. 2004 Aug;40(Pt 1):5-16
references.pmid: 15270702
references.citation: Exp Neurol. 2004 Oct;189(2):280-92
references.pmid: 15380479
references.citation: Biotechnol Bioeng. 2004 Nov 5;88(3):264-72
references.pmid: 15486930
references.citation: Nature. 1975 Nov 27;258(5533):341-2
references.pmid: 1196362
references.citation: Proc Natl Acad Sci U S A. 1976 Jul;73(7):2424-8
references.pmid: 1065897
references.citation: EMBO J. 1992 Mar;11(3):917-22
references.pmid: 1547788
references.citation: Proc Natl Acad Sci U S A. 1994 Apr 12;91(8):3247-51
references.pmid: 8159733
references.citation: Dev Biol. 1995 Apr;168(2):342-57
references.pmid: 7729574
references.citation: Exp Neurol. 1997 Apr;144(2):350-60
references.pmid: 9168835
references.citation: Science. 1999 Jan 15;283(5400):381-7
references.pmid: 9888852
references.citation: J Biol Chem. 1999 Oct 15;274(42):30169-81
references.pmid: 10514507
references.citation: Exp Neurol. 2005 Jan;191(1):65-76
references.pmid: 15589513
references.citation: Dev Growth Differ. 2004 Oct;46(5):393-403
references.pmid: 15606485
references.citation: Sci STKE. 2006 Jan 24;2006(319):pe5
references.pmid: 16434726
references.citation: Ann N Y Acad Sci. 2006 Apr;1068:352-66
references.pmid: 16831937
references.citation: Biomaterials. 2006 Dec;27(36):5990-6003
references.pmid: 16919326
references.citation: Biochem Soc Trans. 2006 Dec;34(Pt 6):1287-90
references.pmid: 17073803
references.citation: Biochem Biophys Res Commun. 2007 Jun 15;357(4):903-9
references.pmid: 17466268
references.citation: Stem Cells. 2007 Sep;25(9):2235-44
references.pmid: 17585170
references.citation: Neurobiol Dis. 2006 Jun;22(3):509-22
references.pmid: 16497507