Failure of Y-27632 to improve the culture of adult human adipose-derived stem cells

Abstract

Y-27632 is a well-known inhibitor of the Rho-associated coiled kinase (ROCK) and has been shown to significantly improve the culture of a variety of multipotent stem cell types. However, the effects of Y-27632 on the expansion of adult human adipose-derived stem cell (hADSC) cultures remain to be established. Here, we aimed to characterize the effects of Y-27632 on the culture of hADSCs. Adult hADSCs were isolated from subjects submitted to elective plastic surgery procedures and cultivated in vitro under optimized conditions. Our results show that the continuous supplementation of hADSC cultures with Y-27632 led to decreased numbers of cells and decreased global metabolic viability of hADSC cultures when compared with control conditions. This effect appeared to be dependent on the continuous presence of the drug and was shown to be concentration-dependent and significant for 10 μM and 20 μM of Y-27632. Moreover, the Y-27632-induced decrease in hADSC numbers was not linked to a block in global cell proliferation, as cell numbers consistently increased from the moment of plating until passaging. In addition, Y-27632 was not able to increase the number of hADSCs present in culture 24 hours after passaging. Taken together, our results suggest that, in contrast to other stem cell types, Y-27632 supplementation is not a suitable strategy to enhance hADSC culture expansion.

Keywords:

• human mesenchymal stem cells
• human multipotent stromal cells (hMSCs)
• ROCK inhibitor
• MTS assay

Acknowledgments

We thank Jeffrey M Gimble (Center for Stem Cell Research and Regenerative Medicine, Tulane University and LaCell LLC, New Orleans, LA, USA) for kindly isolating, characterizing, and sharing the cellular lines of hADSCs used in the present study and for critical input on the manuscript. We also would very much like to thank Laurent Roybon (Stem Cell Laboratory for CNS Disease Modeling, Department of Experimental Medical Science, Lund University, Lund, Sweden) for the utilization of Metamorph NX software for automated cell quantification. We are grateful to Miguel Carvalho, Ana Pires, Eduardo Gomes, Fábio Teixeira and Nuno Silva for technical assistance. This work was supported by the Portuguese Foundation for Science and Technology (predoctoral fellowship to NJL [SFRH/BD/33421/2008]; FCT Investigator Program to AJS) and the Luso-American Development Foundation.

Author contributions

NJL: collection of data, conception and design, data analysis and interpretation, manuscript writing; SCS: collection of data, data analysis and interpretation, manuscript writing; AJS: conception and design, data analysis and interpretation, manuscript writing; NS: conception and design, data analysis and interpretation, manuscript writing. All authors read and approved the final manuscript.

Disclosure

The authors report no conflicts of interest in this work.