Combination of Low Calcium with Y-27632 Rock Inhibitor Increases the Proliferative Capacity, Expansion Potential and Lifespan of Primary Human Keratinocytes while Retaining Their Capacity to Differentiate into Stratified
Authors
Xanthe L. Strudwick, Debbie L. Lang, Louise E. Smith, Allison J. Cowin
Institution
University of South Australia, Mawson Lakes,
Country
Australia
Year
2015
Journal
PLOS One
Abstract
Human keratinocytes are difficult to isolate and have a limited lifespan. Traditionally, immortalised
keratinocyte cell lines are used in vitro due to their ability to bypass senescence and
survive indefinitely. However these cells do not fully retain their ability to differentiate in vitro
and they are unable to form a normal stratum corneum in organotypic culture. Here we
aimed to generate a pool of phenotypically similar keratinocytes from human donors that
could be used in monolayer culture, without a fibroblast feeder layer, and in 3D human skin
equivalent models. Primary human neonatal epidermal keratinocytes (HEKn) were cultured
in low calcium, (0.07mM) media, +/-10μM Y-27632 ROCK inhibitor (HEKn-CaY). mRNA
and protein was extracted and expression of differentiation markers Keratin 14 (K14), Keratin
10 (K10) and Involucrin (Inv) assessed by qRT-PCR and Western blotting. The differentiation
potential of the HEKn-CaY cultures was assessed by increasing calcium levels and
removing the Y-27632 for 72hrs prior to assessment of K14, K10 and Inv. The ability of the
HEKn-CaY, to form a stratified epithelium was assessed using a human skin equivalent
(HSE) model in the absence of Y-27632. Increased proliferative capacity, expansion potential
and lifespan of HEKn was observed with the combination of low calcium and 10μM
ROCK inhibitor Y-27632. The removal of Y-27632 and the addition of high calcium to induce
differentiation allowed the cells to behave as primary keratinocytes even after extended serial
passaging. Prolonged lifespan HEK-CaYs were capable of forming an organised stratified
epidermis in 3D HSE cultures, demonstrating their ability to fully stratify and retain their
original, primary characteristics. In conclusion, the use of 0.07mM Calcium and 10μM
Y-27632 in HEKn monocultures provides the opportunity to culture primary human keratinocytes
without a cell feeder layer for extended periods of culture whilst retaining their ability
to differentiate and form a stratified epithelium.
PLOS