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; h2r107@gmail.com

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