Abstract
Co-culture of human primary epithelial cells with irradiated 3T3 fibroblast feeder cells (J2 cells) and the Rho kinase inhibitor Y-27632 (Y) allows for the unrestricted growth of cells of epithelial origin by the process termed conditional reprogramming. To better understand the nature of the signaling processes associated with conditionally reprogrammed cells, the effect of the two critical components of the co-culture conditions, J2 cells and Y, on the growth of human foreskin keratinocytes (HFKs) was evaluated by gene expression profiling, reverse-phase protein arrays and siRNA screening. J2 cells and Y acted cooperatively to down-regulate differentiation, and upregulate proliferation and cell adhesion, including increased pT308Akt and pERK, and reduced TGF-β pathway signaling. These findings establish a mechanistic basis for the unlimited growth potential of human epithelial cells that will be invaluable to assess the effect of genetic changes in pathologic tissues and their response to therapeutic agents.
Highlights
The capacity of human epidermal keratinocytes for sustained growth depends on the relative cell composition emanating from the basal layer [1,2,3]
Functional siRNA library screening and reverse-phase protein arrays (RPPA), we found that J2 cells and Y produced a cooperative effect on human foreskin keratinocytes (HFKs), which resulted in suppression of TGF-β signaling juxtaposed with up-regulation of changes in cell cycle progression, cell adhesion and motility
The Rho kinase inhibitor Y-27632 has been reported to reduce stress fibers and the actin microarchitecture [15, 16], cells in coculture do not depict its negative effect on cell growth
Summary
The capacity of human epidermal keratinocytes for sustained growth depends on the relative cell composition emanating from the basal layer [1,2,3]. This co-culture technique was modified to include epithelial cells from prostate, breast, trachea, liver and lung, wherein the use of J2 cells and the Rho kinase inhibitor Y-27632 (Y) resulted in the rapid reprogramming of cells into immortalized karyotype-stable cultures [6, 7] These conditionally reprogrammed epithelial cells expressed markers of adult stem cells, but not of embryonic and induced pluripotent stem cells, and appeared to consist of cell populations resembling their primary tissue of origin, making them ideal to study tissue regeneration, as well as normal physiologic and pathologic processes [8]. This technique was employed recently to PLOS ONE | DOI:10.1371/journal.pone.0116755 February 25, 2015
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