Kinetic analysis of neurotrophin-3-mediated differentiation of embryonic stem cells into neurons.
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.
Willerth
Stephanie M
SM
Department of Biomedical Engineering, Washington University, St. Louis, Missouri 63130, USA.
Sakiyama-Elbert
Shelly E
SE
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
D051792:Basic Helix-Loop-Helix Proteins; D048049:Extracellular Signal-Regulated MAP Kinases; D020933:Neurotrophin 3; C499317:Ascl1 protein, mouse
Development. 2006 Mar;133(5):889-900
16452094
Neuroscience. 2008 Jan 2;151(1):138-47
18093741
Neuron. 2005 Jan 20;45(2):189-92
15664170
Bioinformatics. 2005 Mar15;21(6):741-53
15479714
Tissue Eng. 2005 Mar-Apr;11(3-4):506-12
15869429
Exp Cell Res. 2005 Jun 10;306(2):343-8
15925590
Nat Med. 1999 Dec;5(12):1410-2
10581084
Blood. 2000 Aug 15;96(4):1215-22
10942360
FEBS Lett. 2000 Oct 6;482(3):169-74
11024454
Stem Cells. 2002;20(2):119-38
11897869
Nat Biotechnol. 2002 Apr;20(4):370-5
11923843
J Neurosci Res. 2003 Mar 1;71(5):648-58
12584723
J Neurosci. 2003 Jun 15;23(12):5149-60
12832539
J Recept Signal Transduct Res. 2003;23(2-3):197-209
14626447
Biochem Biophys Res Commun. 2004 Feb 20;314(4):1113-20
14751248
Proc Natl Acad Sci U S A. 2004 Mar 2;101(9):2900-5
14978270
Trends Biotechnol. 2004 Apr;22(4):155-6
15104108
Biophys J. 2004 May;86(5):2748-57
15111393
Biotechnol Appl Biochem. 2004 Aug;40(Pt 1):5-16
15270702
Exp Neurol. 2004 Oct;189(2):280-92
15380479
Biotechnol Bioeng. 2004 Nov 5;88(3):264-72
15486930
Nature. 1975 Nov 27;258(5533):341-2
1196362
Proc Natl Acad Sci U S A. 1976 Jul;73(7):2424-8
1065897
EMBO J. 1992 Mar;11(3):917-22
1547788
Proc Natl Acad Sci U S A. 1994 Apr 12;91(8):3247-51
8159733
Dev Biol. 1995 Apr;168(2):342-57
7729574
Exp Neurol. 1997 Apr;144(2):350-60
9168835
Science. 1999 Jan 15;283(5400):381-7
9888852
J Biol Chem. 1999 Oct 15;274(42):30169-81
10514507
Exp Neurol. 2005 Jan;191(1):65-76
15589513
Dev Growth Differ. 2004 Oct;46(5):393-403
15606485
Sci STKE. 2006 Jan 24;2006(319):pe5
16434726
Ann N Y Acad Sci. 2006 Apr;1068:352-66
16831937
Biomaterials. 2006 Dec;27(36):5990-6003
16919326
Biochem Soc Trans. 2006 Dec;34(Pt 6):1287-90
17073803
Biochem Biophys Res Commun. 2007 Jun 15;357(4):903-9
17466268
Stem Cells. 2007 Sep;25(9):2235-44
17585170
Neurobiol Dis. 2006 Jun;22(3):509-22
16497507