Improving Viability and Transfection Efficiency with Human Umbilical Cord Wharton\\\'s Jelly Cells Through Use of a ROCK Inhibitor

Authors:
Mellott AJ, Godsey ME, Shinogle HE, Moore DS, Forrest ML, Detamore MS.
In:
Source: Cell Rep
Publication Date: (2014)
Issue: 16 (2): 91-7
Research Area:
Stem Cells
Gene Expression
Cells used in publication:
Mesenchymal stem cell (MSC), human
Species: human
Tissue Origin: bone marrow
Culture Media:
Platform:
4D-Nucleofector™ X-Unit
Experiment
5x10e5 cells, -solution P1, 100µl cuvette, -program FF-104, -5µg pmaxGFP pre and post nucleofection culture with ROCK inhibitor
Abstract
Differentiating stem cells using gene delivery is a key strategy in tissue engineering and regenerative medicine applications. Nonviral gene delivery bypasses several safety concerns associated with viral gene delivery; however, leading nonviral techniques, such as electroporation, subject cells to high stress and can result in poor cell viabilities. Inhibition of Rho-associated coiled-coil kinase (ROCK) has been shown to mitigate apoptotic mechanisms associated with detachment and freezing of induced pluripotent stem cells and embryonic stem cells; however, inhibiting ROCK in mesenchymal stromal cells (MSCs) for improving gene delivery applications has not been reported previously. In this study, we hypothesized that ROCK Inhibitor (RI) would improve cell viability and gene expression in primary human umbilical cord mesenchymal stromal cells (hUCMSCs) when transfected via Nucleofection™. As hypothesized, the pre-treatment and post-treatment of hUCMSCs transfected via nucleofection with Y-27632-RI significantly improved survival rates of hUCMSCs and gene expression as measured by green fluorescent protein intensity. This study provides the first comparative look at the effect of Y-27632-RI on hUCMSCs that underwent transfection via nucleofection and shows that using Y-27632-RI in concert with nucleofection could greatly enhance the utility of differentiating and reprogramming hUCMSCs for tissue engineering applications.