Are the Effects of the Cholera Toxin and Isoproterenol on Human Keratinocytes’ Proliferative Potential Dependent on Whether They Are Co-Cultured with Human or Murine Fibroblast Feeder Layers?

Abstract

Human keratinocyte culture has provided the means to treat burns, wounds and skin pathologies. To date, to efficiently culture keratinocytes, cells are cultured on an irradiated feeder layer (iFL), either comprising human (iHFL) or murine (i3T3FL) fibroblasts, and the culture medium is supplemented with a cyclic adenosine monophosphate (cAMP) accumulation inducing agent such as isoproterenol (ISO) or cholera toxin (CT). Previous studies have characterized how the feeder layer type and the cAMP inducer type influence epithelial cells’ phenotype independently from one another, but it is still unknown if an optimal combination of feeder layer and cAMP inducer types exists. We used sophisticated statistical models to search for a synergetic effect of feeder layer and cAMP inducer types on human keratinocytes’ proliferative potential. Our data suggests that, when culturing human keratinocytes, using iHFL over i3T3FL increases population doublings and colony-forming efficiency through signaling pathways involving Ak mouse strain thymoma (Akt, also known as protein kinase B) isoforms 1 to 3, signal transducer and activator of transcription 5 (STAT5), p53, and adenosine monophosphate activated protein kinase α1 (AMPKα1). Both tested cAMP inducers ISO and CT yielded comparable outcomes. However, no significant synergy between feeder layer and cAMP inducer types was detected. We conclude that, to promote human keratinocyte growth in the early passages of culture, co-culturing them with a human feeder layer is preferable to a murine feeder layer.

A Tissue-Engineered Human Psoriatic Skin Model to Investigate the Implication of cAMP in Psoriasis: Differential Impacts of Cholera Toxin and Isoproterenol on cAMP Levels of the Epidermis

“…Indeed, CT was the strongest agent in affecting cAMP concentration among the four different cAMP stimulators tested [15]. Moreover, these results support the use of both cAMP inducers, at appropriate concentrations, when producing healthy reconstructed skin models, and are in accord with results reported by Cortez Ghio et al [38]. Isoproterenol (ISO) is therefore a good candidate for replacing CT since it presents several advantages regarding safety and regulations [38].…”

“…Moreover, these results support the use of both cAMP inducers, at appropriate concentrations, when producing healthy reconstructed skin models, and are in accord with results reported by Cortez Ghio et al [38]. Isoproterenol (ISO) is therefore a good candidate for replacing CT since it presents several advantages regarding safety and regulations [38]. Indeed, CT is known to be potentially toxic and therefore requires more precautions in its manipulation [39,40].…”

The Human Tissue-Engineered Cornea (hTEC): Recent Progress

“…We recently substituted i3T3 with a new irradiated human dermal fibroblast feeder layer [ 190 ]. This particular feeder layer has proven to be effective for culturing keratinocytes [ 191 , 192 ] and has even yielded higher growth rates and increased clonogenicity when compared with i3T3 [ 193 ]. We recently replaced cholera toxin with isoproterenol, another cAMP inducer whose use in keratinocyte culture goes back to 2005 [ 194 ].…”

“…It therefore has the advantage of being already approved for the clinic. While the measure of its relative effectiveness over cholera toxin has not reached a consensus yet [ 195 , 196 ], we recently demonstrated that isoproterenol was as effective as cholera toxin for stimulating and maintaining the proliferative potential of human skin keratinocytes upon cell passages, without, however, having any restrictions related to its use [ 193 ]. As for limbal epithelial cells, isoproterenol also proved to be more effective than cholera toxin for inducing cell proliferation, as well as for maintaining a smaller cell size [ 197 ].…”