fate manipulation of pc-12 cell using ... manipulation of pc-12 cell using microfluidic device...
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FATE MANIPULATION OF PC-12 CELL USING MICROFLUIDIC DEVICE Hyunryul Ryu1*, Minhwan Chung1*, Sung Sik Lee2, Noo Li Jeon1, and Olivier Pertz3 1School of Mechanical and Aerospace Engineering, Seoul National University, KOREA.
2Institute of Biochemistry, ETH Zurich, SWITZERLAND. 3Department of Biomedicine, University of Basel, SWITZERLAND.
*Both authors contribute equally. ABSTRACT
Duration of the ERK activity has been proposed to control PC-12 cell fate. In this research, with integrated platform
of FRET sensor and Microfluidic pulse generator, we tested the single cellular response of the ERK signaling pathway to
the external patterned growth factor stimulations. With sustained stimulation of the growth factor gives the well-known
activity curves in average; transient in EGF and sustained in NGF. However, we found that the pulsed stimulation of the
growth factor can not only re-trigger the pathway, but also could manipulate the fate of the PC-12 cell.
KEYWORDS: FRET, Microfluidics, pulsed stimulation, differentiation
INTRODUCTION
The extracellular-signal regulated kinases (ERK) 1 and 2 are known as the key molecule to determine the cell fate;
differentiation or proliferation. In PC-12 cells, epidermal growth factor (EGF) triggers the transient response of the ERK,
which induces cell proliferation. On the other hand, the sustained activation of the molecule induced by neural growth
factor (NGF), which is known as the determinant of differentiation. Duration of the ERK activity has thus been proposed
to control PC-12 cell fate.
THEORY
Although both NGF and EGF activates ERK molecules in PC12 cells the same, the results are totally different;
differentiation and proliferation. It is likely coming from the kinetics of the ERK molecule. One possible hypothesis is
the immediate early gene(IEG) that induces differentiation of PC-12 is stabilized by NGF thus by ERK, not by EGF. To
prove this , we set up the theory; multiple pulse of the EGF, which can re-trigger ERK, can behave as the sustained ERK
level of the NGF stimulation so that PC-12 can be differentiated.
EXPERIMENT
Figure 1. (A) Schematic diagram of the Bio-
sensor, EKAR2G1 and (B) microfluidic platform
for pulsatile stimulation of the growth factor. (C)
The Average Emission ratio from the entire pop-
ulation of each sustained growth factor experi-
ment. Plot gives the same result that the conven-
tional methods used to give. (D,E) Also, Ultra-
sensitivity, which is the characteristic property of
the ERK pathway, was observed. However, fol-
lowing single cellular response of the ERK was
shown different response. They showed three
identical curve phenotype; sustained, oscillated,
and transient kinetics in both EGF stimulation
(F,H) and NGF stimulation (G,I).
978-0-9798064-6-9/µTAS 2013/$20©13CBMS-0001 1911 17th International Conference on MiniaturizedSystems for Chemistry and Life Sciences27-31 October 2013, Freiburg, Germany
To get the reproducible results from the ERK sensor, we transfect the biosensor, named EKAR2G1, stably expressing
in the PC-12 cell-line. On the other hand, PC-controlled pressure pump was connected to the high-throughput
microfluidic pulse generators to control the precise temporal pulse stimulation of the growth factors. This platform
enables us to get a large amount of single cell trajectories, and precise control of the GF concentration. After the
experiments, image analysis was done automatically by the FIJI-based macro. This sequences of the experiments let us
make a large amount of database about the ERK pathway of individual cell in a short time. To make sure the reliability
of the assay, we correlated the results with western blot and immuno-stainining.,
RESULTS AND DISCUSSION
As shown in Figure 1, the average of ERK activation level seemed the same as traditional methods, sustained in NGF
and transient in EGF. However, single cell trajectories showed all transient, oscillatory, and sustained curve
phenotypes in both GF stimulations. The different average behavior between NGF and EGF stimulation was seemed to
be come from the different proportion of the each phenotype; more transient curves in EGF and more sustained ones in
NGF.
Meanwhile, the pulsed stimulation of both GFs made synchronized transient ERK response over the population. With
multiple pulse stimulation, we identify that a certain frequency of pulses could retrigger the system for a proper amount
of time to mimic sustained activation of the pathway. We could differentiate cells with multiple pulsed EGF as much as
sustained NGF. Furthermore, multiple pulsed NGF increases differentiation ratio up to 20%. These results propose that
that kinetics of the ERK activity could be the key of the cell-fate decision.
CONCLUSION
The result suggests that the mimic of the MAP-Kinase activity by changing extracellular environment can manipulate
not only the signal transduction of the pathway, but also the cell fate. We showed that the retriggered pERK could stabi-
lize the immediate early gene, enough to differentiate the cell.
Figure 2. The real-time activity of the ERK molecules, stimulated by various types of stimulation pat-
terns.
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Figure 3. (A) Plots of Differentiation ratio and (B) neurite length clearly shows that the manipulation of ERK activity
can induce the different cell fate, regardless of GFs. (C) Images of PC-12 with various types of stimulation patterns.
ACKNOWLEDGEMENTS
This work was supported by WCU (World Class University) program (R31-2008-000-10083-0) through the National
Research Foundation funded by the Ministry of Education, Science and Technology of Korea.
REFERENCES
[1] S. D. M. Santos, P. J. Verveer, P. I. H. Bastiaens, “Growth factor-induced MAPK network topology shapes Erk re-
sponse determining PC-12 cell fate,” Nature 9, 324
[2] J.-Y. Chen, J.-R. Lin, K. A. Cimprich, T. Meyer, “A two-dimensional ERK-AKT signaling code for an NGF-
triggered cell-fate decision,” Molecular Cell 45, 196
[3] L. O. Murphy, S. Smith, R. Chen, D. C. Finger, J. Blenis, “Molecular interpretation of ERK signal duration by im-
mediate early gene products,” Nature Cell Biology 4, 555
CONTACT
*H. Ryu, tel: +82-2-880-1646; [email protected]
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