Effect of ROCK Inhibitor Y-27632 on Normal and Variant Human Embryonic Stem Cells (hESCs) in Vitro: Its Benefits in HESC Expansion

Overview

Journal Stem Cell Rev Rep

Publisher Springer

Specialty Cell Biology

Date 2009 Dec 17

PMID 20013076

Citations 37

Authors

Kalamegam Gauthaman

Chui-Yee Fong

Ariff Bongso

Affiliations

Soon will be listed here.

Abstract

The Rho associated coiled coil protein kinase (ROCK) dependent signaling pathway plays an important role in numerous physiological functions such as cell proliferation, adhesion, migration and inflammation. Human embryonic stem cells (hESCs) undergo differentiation and poor survival after single cell dissociation in culture thus limiting their expansion for cell based therapies. We evaluated the role of the selective ROCK inhibitor Y-27632 on hESC colonies and disassociated single hESCs from two different hESC lines. Karyotypically normal hESCs (HES3) and variant hESCs (BG01V) were treated with Y-27632 at 5, 10 and 20 muM concentrations for 72 h and its effects on hESC self renewal, colony morphology, cell cycle and pluripotency were evaluated. Increased cell proliferation of both HES3 and BG01V were observed for all three concentrations compared to untreated controls following passaging of cell clusters or dissociated single cells and some of these increases were statistically significant. Cell cycle assay demonstrated normal cell cycle progression with no peaks evident of apoptosis. No morphological differentiation was evident following treatment with the highest concentration of Y-27632 (20 muM) and the stemness related genes continued to be highly expressed in both HES3 and BG01V cells compared to untreated controls. The results confirmed that Y-27632 is a useful agent that aids in the expansion of undifferentiated hESC numbers for downstream applications in regenerative medicine.

Citing Articles

Improvement of post-thaw quality and fertilizing ability of bull spermatozoa using Rho kinase inhibitor in freezing extender.

Behnam M, Asadpour R, Rezaei Topraggaleh T, Hamali H Front Vet Sci. 2023; 10:1155048.

PMID: 37483290 PMC: 10359164. DOI: 10.3389/fvets.2023.1155048.

A Cell-Based Optimised Approach for Rapid and Efficient Gene Editing of Human Pluripotent Stem Cells.

Cuevas-Ocana S, Yang J, Aushev M, Schlossmacher G, Bear C, Hannan N Int J Mol Sci. 2023; 24(12).

PMID: 37373413 PMC: 10299534. DOI: 10.3390/ijms241210266.

Early Differentiation Signatures in Human Induced Pluripotent Stem Cells Determined by Non-Targeted Metabolomics Analysis.

Abdalkader R, Chaleckis R, Fujita T Metabolites. 2023; 13(6).

PMID: 37367864 PMC: 10301689. DOI: 10.3390/metabo13060706.

Flavonoids from exerts protective effect on ischemia-induced cerebral injury in mice via inhibiting RhoA-ROCK2 signaling pathway.

Zheng Y, Lin X, Ren M, Song K, Chen Y, Zeng L Zhejiang Da Xue Xue Bao Yi Xue Ban. 2023; 52(1):77-87.

PMID: 37283121 PMC: 10407990. DOI: 10.3724/zdxbyxb-2022-0567.

Development and characterization of ferret tracheal injury and cell engraftment model.

Ievlev V, Pai A, Dillon Jr D, Kuhl S, Lynch T, Freischlag K Front Med (Lausanne). 2023; 10:1144754.

PMID: 37113613 PMC: 10126424. DOI: 10.3389/fmed.2023.1144754.

References

Okita K, Ichisaka T, Yamanaka S . Generation of germline-competent induced pluripotent stem cells. Nature. 2007; 448(7151):313-7. DOI: 10.1038/nature05934. View

Sahai E, Marshall C . ROCK and Dia have opposing effects on adherens junctions downstream of Rho. Nat Cell Biol. 2002; 4(6):408-15. DOI: 10.1038/ncb796. View

Harb N, Archer T, Sato N . The Rho-Rock-Myosin signaling axis determines cell-cell integrity of self-renewing pluripotent stem cells. PLoS One. 2008; 3(8):e3001. PMC: 2500174. DOI: 10.1371/journal.pone.0003001. View

Bishop A, Hall A . Rho GTPases and their effector proteins. Biochem J. 2000; 348 Pt 2:241-55. PMC: 1221060. View

Emgard M, Hallin U, Karlsson J, Bahr B, Brundin P, Blomgren K . Both apoptosis and necrosis occur early after intracerebral grafting of ventral mesencephalic tissue: a role for protease activation. J Neurochem. 2003; 86(5):1223-32. DOI: 10.1046/j.1471-4159.2003.01931.x. View

Shim J, Kim S, Woo D, Kim S, Oh C, McKay R . Directed differentiation of human embryonic stem cells towards a pancreatic cell fate. Diabetologia. 2007; 50(6):1228-38. DOI: 10.1007/s00125-007-0634-z. View

Reubinoff B, Pera M, Fong C, Trounson A, Bongso A . Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol. 2000; 18(4):399-404. DOI: 10.1038/74447. View

Raftopoulou M, Hall A . Cell migration: Rho GTPases lead the way. Dev Biol. 2003; 265(1):23-32. DOI: 10.1016/j.ydbio.2003.06.003. View

Laflamme M, Chen K, Naumova A, Muskheli V, Fugate J, Dupras S . Cardiomyocytes derived from human embryonic stem cells in pro-survival factors enhance function of infarcted rat hearts. Nat Biotechnol. 2007; 25(9):1015-24. DOI: 10.1038/nbt1327. View

10.

Sottile V, Thomson A, McWhir J . In vitro osteogenic differentiation of human ES cells. Cloning Stem Cells. 2003; 5(2):149-55. DOI: 10.1089/153623003322234759. View

11.

Riento K, Ridley A . Rocks: multifunctional kinases in cell behaviour. Nat Rev Mol Cell Biol. 2003; 4(6):446-56. DOI: 10.1038/nrm1128. View

12.

Maruta H, Nheu T, He H, Hirokawa Y . Rho family-associated kinases PAK1 and rock. Prog Cell Cycle Res. 2003; 5:203-10. View

13.

Ishizaki T, Naito M, Fujisawa K, Maekawa M, Watanabe N, Saito Y . p160ROCK, a Rho-associated coiled-coil forming protein kinase, works downstream of Rho and induces focal adhesions. FEBS Lett. 1997; 404(2-3):118-24. DOI: 10.1016/s0014-5793(97)00107-5. View

14.

Martin-Ibanez R, Unger C, Stromberg A, Baker D, Canals J, Hovatta O . Novel cryopreservation method for dissociated human embryonic stem cells in the presence of a ROCK inhibitor. Hum Reprod. 2008; 23(12):2744-54. DOI: 10.1093/humrep/den316. View

15.

Bongso A, Fong C, Gauthaman K . Taking stem cells to the clinic: Major challenges. J Cell Biochem. 2008; 105(6):1352-60. DOI: 10.1002/jcb.21957. View

16.

Thomson J, Itskovitz-Eldor J, Shapiro S, Waknitz M, Swiergiel J, Marshall V . Embryonic stem cell lines derived from human blastocysts. Science. 1998; 282(5391):1145-7. DOI: 10.1126/science.282.5391.1145. View

17.

Mummery C, Ward D, van den Brink C, Bird S, Doevendans P, Opthof T . Cardiomyocyte differentiation of mouse and human embryonic stem cells. J Anat. 2002; 200(Pt 3):233-42. PMC: 1570681. DOI: 10.1046/j.1469-7580.2002.00031.x. View

18.

Pirone D, Liu W, Alom Ruiz S, Gao L, Raghavan S, Lemmon C . An inhibitory role for FAK in regulating proliferation: a link between limited adhesion and RhoA-ROCK signaling. J Cell Biol. 2006; 174(2):277-88. PMC: 2064187. DOI: 10.1083/jcb.200510062. View

19.

Cheng Q, Sasaki Y, Shoji M, Sugiyama Y, Tanaka H, Nakayama T . Cdk5/p35 and Rho-kinase mediate ephrin-A5-induced signaling in retinal ganglion cells. Mol Cell Neurosci. 2003; 24(3):632-45. DOI: 10.1016/s1044-7431(03)00220-3. View

20.

Hornstein E, Benvenisty N . The "brainy side" of human embryonic stem cells. J Neurosci Res. 2004; 76(2):169-73. DOI: 10.1002/jnr.20034. View