Establishment of an EB keratinocyte collection using Rho kinase chemical transformation (McLean-McGrath 1)Completed
|Project lead||Prof Irwin McLean & Prof John McGrath|
|Organisation||University of Dundee School of Medicine, Ninewells Hospital and Medical School|
|Project budget||GBP 67,007.00|
|Start date / Duration||01. Oct 2011 / 12 months|
|Funder(s) / Co-Funder(s)||DEBRA UK, MSAP/EBEP Recommended|
|Research area||Molecular therapy|
Short lay summary
Current methods to immortalize keratinocytes lead to genomic instability which is obviously undesirable. The Rho kinase (ROCK) inhibitor Y-27632 has been shown to extend the lifespan of keratinocytes essentially indefinitely, with genomic stability and retention of differentiation potential. The drawback is the cost (~€1200 per litre of culture medium). Dundee Drug Discovery Unit has developed a range of new, cheap ROCK inhibitors, such as DDD00033325. Here, we will test the best of these with comparison to Y-27632 for their ability to immortalize keratinocytes from EB patients and controls. These ROCK Inhibitor Transformed (ROCKIT) keratinocytes will be subjected to tests of genomic stability and differentiation potential and a protocol developed for routine use. A bank of ROCKIT cell lines will be developed from EB patients with a range of different mutations in the 14 EB causative genes, which will be distributed to researchers in the field. This work will provide a useful resource for the future study of EB pathomechanisms as well as therapy development.
The overall aim of this 24 month grant proposal was to develop a routine and cost effective method for producing long-term keratinocyte cell lines using a novel Rho kinase inhibitor (also known as ROCK inhibitor), ensure these were genetically “normal”, able to form 3D cultures and ultimately, to exploit this technology to produce banks of cultures able to support basic physiological studies and therapeutic developments in EB.
What did this project achieve?
- The research group found that Rho kinase inhibitors can prolong the life-span of primary keratinocytes allowing the generation of larger numbers of cells without resorting to transformation.
- The in-house Rho kinase inhibitor DDD33325 was only modestly able to extend keratinocyte culture lifespan (up to 17 passages), whereas the commercial inhibitor Y27632 was able to significantly increase culture longevity (>25 passages).
- Although all three ROCK inhibitors were shown to have similar kinase inhibtory profiles, it is possible that Y27632 has some other additional specificity not tested for that makes this compound more effective at extending culture lifespan.
- The team was happy to distribute samples of DDD33325 to any research groups who want to try this compound.
- They identified 5 further ROCK inhibitor compounds with further improved potency for future studies.
- The research team found that the ability of cells treated with Rho kinase inhibitors to form 3D differentiated cultures was very poor, which limits downstream applications of these conditionally transformed cultures.
- Therefore, despite the initial promise of developing a bank of an EB keratinocyte collection, the results obtained in the first 12 months of this grant proposal showed poor prospects of successful development and it was therefore decided to end this project early, after 12 months.