Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
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Improvement of Cell Viability Using a Rho-associated Protein Kinase (ROCK) Inhibitor in Human Dental Papilla derived Single-induced Pluripotent Stem Cells

ROCK 억제제를 통한 사람 치유두 조직 유래 단일 사람 유도만능줄기세포의 생존성 향상

  • Shim, Yoo-Jin (Department of Biology Education, Gyeongsang National University) ;
  • Kang, Young-Hoon (Department of Oral and Maxillofacial Surgery, Gyeongsang National University) ;
  • Kim, Hyeon-Ji (Department of Biology Education, Gyeongsang National University) ;
  • Kim, Mi-Jeong (Department of Biology Education, Gyeongsang National University) ;
  • Lee, Hyeon-Jeong (OBS/Theriogenology and Biotechnology, Gyeongsang National University) ;
  • Son, Young-Bum (OBS/Theriogenology and Biotechnology, Gyeongsang National University) ;
  • Lee, Sung-Ho (Division of Life Science, Gyeongsang National University) ;
  • Jeon, Byeong-Gyun (Department of Biology Education, Gyeongsang National University)
  • Received : 2019.06.03
  • Accepted : 2019.07.11
  • Published : 2019.08.30
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Abstract

The aim of the present study was to improve the cell viability of human dental papilla derived single-induced pluripotent stem cells (iPSCs) using a Rho-associated protein kinase (ROCK) inhibitor, Y-27632. The iPSCs were produced using an episomal plasmid-based reprogramming method. After cell separation using trypsin, the iPSCs were treated with 0, 0.5, 1, 2.5, 5, 7.5, or $10{\mu}M$ Y-27632 for 5 d. Cell viability increased significantly following the $5{\mu}M$ Y-27632 treatment (p<0.05). When the iPSCs were exposed to medium containing $10{\mu}M$ Y-27632 for 0, 1, 2, 3, 4, and 5 d, the cell viability rate increased significantly in accordance with the cell viability rate (p<0.05). To evaluate the effect of the Y-27632 treatment on stemness characteristics, the expression of stem cell-specific transcripts and telomerase activity were investigated in the iPSCs treated with $10{\mu}M$ Y-27632 for 5 d. The expression levels of stem cell-specific transcripts, such as OCT-4, NONOG, and SOX-2, and telomerase activity were not significantly different in the iPSCs treated with $10{\mu}M$ Y-27632 as compared with those of untreated control iPSCs (p>0.05). Taken together, the results demonstrated that cell viability can be improved by treatment with the ROCK inhibitor Y-27632, without losing iPSC stemness characteristics.

이 연구는 단일 세포로 분리된 유도만능줄기세포(induced pluripotent stem cells, iPSCs)에 anoikis 세포사멸을 억제할 수 있는 Rho-associated protein kinase (ROCK)의 억제제를 처리하여 iPSCs의 세포 생존성을 향상하고자 하였다. Episomal plasmid 방법으로 확립된 iPSCs를 단일세포로 분리한 후, ROCK 억제제 Y-27632 dihydrochloride (Y-27632)를 0 uM, 0.5 uM, 1 uM, 2.5 uM, 5 uM, 7.5 uM 및 10 uM 농도별로 5주일 동안 각각 처리하였을 때, 5 uM 이상의 농도에서 세포의 생존율이 유의적으로 향상되었고, 10 uM의 Y-27632을 0일, 1일, 2일, 3일, 4일 및 5일 동안 처리하였을 때, Y-27632의 노출 기간이 길어질수록 세포의 생존율이 유의적으로 향상되는 것을 관찰하였다. 그러나, Y-27632의 노출 후, iPSCs의 형태학적 분화가 관찰되어 10 uM의 Y-27632에서 5일 동안 iPSCs에 처리 한 후, 줄기세포학적인 특성을 비교 조사하였다. 우선, octamer-binding transcription factor 4 (OCT-4), homeobox protein NANOG (NONOG) 및 SRY-box 2 (SOX-2) 줄기세포 특이 유전자의 발현은 Y-27632를 처리한 실험군은 Y-27632를 처리하지 않은 대조군에서 서로 유의적인 차이를 나타내지 않았다. 또한, Y-27632를 처리한 실험군은 Y-27632를 처리하지 않은 대조군과 비교하여 telomerase 활성과 이것의 활성과 관련된 telomerase reverse transcriptase (TERT) 및 telomerase RNA component (TERC)의 유전자 발현에는 유의적인 차이가 없었다. 이상의 결과로 보아, iPSCs에 Y-27632를 처리하였을 때, iPSCs의 줄기세포의 특정을 유지하면서 anoikis에 의한 세포사멸을 감소시켜 세포 생존율이 증가한다는 것을 알 수 있었다.

Keywords

References

  1. Allsopp, R. 2012. Telomere length and iPSC re-programming: survival of the longest. Cell Res. 22, 614-615. https://doi.org/10.1038/cr.2012.6
  2. Amit, M., Carpenter, M. K., Inokuma, M. S., Chiu, C. P., Harris, C. P., Waknitz, M. A., Itskovitz-Eldor, J. and Thomson, J. A. 2000. Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture. Dev. Biol. 227, 271-278. https://doi.org/10.1006/dbio.2000.9912
  3. Beers, J., Gulbranson, D. R., George, N., Siniscalchi, L. I., Jones, J., Thomson, J. A. and Chen, G. 2012. Passaging and colony expansion of human pluripotent stem cells by enzyme-free dissociation in chemically defined culture conditions. Nat. Protoc. 7, 2029-2040. https://doi.org/10.1038/nprot.2012.130
  4. Capetian, P., Azmitia, L., Pauly, M. G., Krajka, V., Stengel, F., Bernhardi, E. M., Klett, M., Meier, B., Seibler, P., Stanslowsky, N., Moser, A., Knopp, A., Gillessen-Kaesbach, G., Nikkhah, G., Wegner, F., Dobrossy, M. and Klein, C. 2016. Plasmid-based generation of induced neural stem cells from adult human fibroblasts. Front. Cell Neurosci. 10, 245.
  5. Gilmore, A. P. 2005. Anoikis. Cell Death Differ. 12 Suppl 2, 1473-1277. https://doi.org/10.1038/sj.cdd.4401723
  6. Ha, H. Y., Jang, S. H. and Jung, J. W. 2011. The use of pluripotent stem cell for personalized cell therapies against neurological disorders. J. Biomed. Biotechnol. 2011, 520816.
  7. Heng, B. C., Ye, C. P., Liu, H., Toh, W. S., Rufaihah, A. J., Yang, Z., Bay, B. H., Ge, Z., Ouyang, H. W., Lee, E. H. and Cao, T. 2006. Loss of viability during freeze-thaw of intact and adherent human embryonic stem cells with conventional slow-cooling protocols is predominantly due to apoptosis rather than cellular necrosis. J. Biomed. Sci. 13, 433-445. https://doi.org/10.1007/s11373-005-9051-9
  8. Huh, Y. J. and Kim, D. W. 2011. Stem cells: general information and perspectives. J. Kor. Med. Assoc. 54, 450-453. https://doi.org/10.5124/jkma.2011.54.5.450
  9. Itoh, K., Yoshioka, K., Akedo, H., Uehata, M., Ishizaki, T. and Narumiya, S. 1999. An essential part for Rho-associated kinase in the transcellular invasion of tumor cells. Nat. Med. 5, 221-225. https://doi.org/10.1038/5587
  10. Jeon, B. G., Jang, S. J., Park, J. S., Subbarao, R. B., Jeong, G. J., Park, B. W. and Rho, G. J. 2015. Differentiation potential of mesenchymal stem cells isolated from human dental tissues into non-mesodermal lineage. Anim. Cell. Syst. 19, 321-331. https://doi.org/10.1080/19768354.2015.1087430
  11. Kim, Y. D., Jang, S. J., Lim, E. J., Ha, J. S., Shivakumar, S. B., Jeong, G. J., Rho, G. J. and Jeon, B. G. 2017. Induction of telomere shortening and cellular apoptosis by sodium meta-arsenite in human cancer cell lines. Anim. Cell. Sys. 21, 241-254. https://doi.org/10.1080/19768354.2017.1342691
  12. Kureishi, Y., Kobayashi, S., Amano, M., Kimura, K., Kanaide, H., Nakano, T., Kaibuchi, K. and Ito, M. 1997. Rho-associated kinase directly induces smooth muscle contraction through myosin light chain phosphorylation. J. Biol. Chem. 272, 12257-12260. https://doi.org/10.1074/jbc.272.19.12257
  13. Lee, H. J. and Jeon, B. G. 2018. Comparison of telomere length and telomerase activity in human normal and cancer cell lines. Contemporary Educational Research 30, 185-200.
  14. Li, X., Meng, G., Krawetz, R., Liu, S. and Rancourt, D. E. 2008. The ROCK inhibitor Y-27632 enhances the survival rate of human embryonic stem cells following cryopreservation. Stem Cells Dev. 17, 1079-1085. https://doi.org/10.1089/scd.2007.0247
  15. Li, X., Meng, G., Krawetz, R., Liu, S. and Rancourt, D. E. 2009. ROCK inhibitor improves survival of cryopreserved serum/feeder-free single human embryonic stem cells. Hum. Reprod. 24, 580-589. https://doi.org/10.1093/humrep/den404
  16. Little, D., Ketteler, R., Gissen, P. and Devine, M. J. 2019. Using stem cell-derived neurons in drug screening for neurological diseases. Neurobiol. Aging 78, 130-141. https://doi.org/10.1016/j.neurobiolaging.2019.02.008
  17. Maldonado, M., Luu, R. J., Ramos, M. E. and Nam, J. 2016. ROCK inhibitor primes human induced pluripotent stem cells to selectively differentiate towards mesendodermal lineage via epithelial-mesenchymal transition-like modulation. Stem Cell Res. 17, 222-227. https://doi.org/10.1016/j.scr.2016.07.009
  18. Matsuoka, T. and Yashiro, M. 2014. Rho/ROCK signaling in motility and metastasis of gastric cancer. World J. Gastroenterol. 20, 13756-12766. https://doi.org/10.3748/wjg.v20.i38.13756
  19. Noritake, J., Watanabe, T., Sato, K., Wang, S. and Kaibuchi, K. 2005. IQGAP1: a key regulator of adhesion and migration. J. Cell Sci. 118(Pt 10), 2085-2092. https://doi.org/10.1242/jcs.02379
  20. Olson, M. F. and Sahai, E. 2008. The actin cytoskeleton in cancer cell motility. Clin. Exp. Metastasis 26, 273-287. https://doi.org/10.1007/s10585-008-9174-2
  21. Olson, M. F. 2008. Applications for ROCK kinase inhibition. Curr. Opin. Cell Biol. 20, 242-248. https://doi.org/10.1016/j.ceb.2008.01.002
  22. Pakzad, M., Totonchi, M., Taei, A., Seifinejad, A., Hassani, S. N. and Baharvand, H. 2010. Presence of a ROCK inhibitor in extracellular matrix supports more undifferentiated growth of feeder-free human embryonic and induced pluripotent stem cells upon passaging. Stem Cell Rev. 6, 96-107. https://doi.org/10.1007/s12015-009-9103-z
  23. Pyle, A. D., Lock, L. F. and Donovan, P. J. 2006. Neurotrophins mediate human embryonic stem cell survival. Nat. Biotechnol. 24, 344-350. https://doi.org/10.1038/nbt1189
  24. Sathananthan, H., Pera, M. and Trounsin, A. 2002. The fine structure of human embryonic stem cells. Reprod. Biomed. Online 4, 56-61. https://doi.org/10.1016/S1472-6483(10)61916-5
  25. Schmidt, R. and Plath, K. 2012. The roles of the reprogramming factors Oct4, Sox2 and Klf4 in resetting the somatic cell epigenome during induced pluripotent stem cell generation. Genome Biol. 13, 251. https://doi.org/10.1186/gb-2012-13-10-251
  26. Thumkeo, D., Watanabe, S. and Narumiya, S. 2013. Physiological roles of Rho and Rho effectors in mammals. Eur. J. Cell Biol. 92, 303-315. https://doi.org/10.1016/j.ejcb.2013.09.002
  27. Walker, A., Su, H., Conti, M. A., Harb, N., Adelstein, R. S. and Sato, N. 2010. Non-muscle myosin II regulates survival threshold of pluripotent stem cells. Nat. Commun. 1, 71. https://doi.org/10.1038/ncomms1074
  28. Watanabe, K., Ueno, M., Kamiya, D., Nishiyama, A., Matsumura, M., Wataya, T., Takahashi, J. B., Nishikawa, S., Muguruma, K. and Sasai, Y. 2007. A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat. Biotechnol. 25, 681-686. https://doi.org/10.1038/nbt1310
  29. Xu, R. H., Peck, R. M., Li, D. S., Feng, X., Ludwing, T. and Thomson, J. A. 2005. Basic FGF and suppression of BMP signaling sustain undifferentiated proliferation of human ES cells. Nat. Methods 2, 185-190. https://doi.org/10.1038/nmeth744
  30. Yamanaka, S. 2012. Induced pluripotent stem cells: past, present, and future. Cell Stem Cell. 10, 678-684. https://doi.org/10.1016/j.stem.2012.05.005
  31. Zhong, X. and Rescorla, F. J. 2012. Cell surface adhesion molecules and adhesion-initiated signaling: understanding of anoikis resistance mechanisms and therapeutic opportunities. Cell Signal. 24, 393-401. https://doi.org/10.1016/j.cellsig.2011.10.005