Long-Term In Vitro Expansion of Epithelial Stem Cells Enabled by Pharmacological Inhibition of PAK1-ROCK-Myosin II and TGF-β Signaling

Abstract

SUMMARYDespite substantial self-renewal capability in vivo, epithelial stem and progenitor cells located in various tissues expand for a few passages in vitro in feeder-free condition before they succumb to growth arrest. Here, we describe the EpiX method, which utilizes small molecules that inhibit PAK1-ROCK-Myosin II and TGF-β signaling to achieve over one trillion-fold expansion of human epithelial stem and progenitor cells from skin, airway, mammary, and prostate glands in the absence of feeder cells. Transcriptomic and epigenomic studies show that this condition helps epithelial cells to overcome stresses for continuous proliferation. EpiX-expanded basal epithelial cells differentiate into mature epithelial cells consistent with their tissue origins. Whole-genome sequencing reveals that the cells retain remarkable genome integrity after extensive in vitro expansion without acquiring tumorigenicity. EpiX technology provides a solution to exploit the potential of tissue-resident epithelial stem and progenitor cells for regenerative medicine.

When the Search for Stemness Genes Meets the Skin Substitute Bioengineering Field: KLF4 Transcription Factor under the Light

“…Considering this demand of regulatory agencies, the substitution of undefined culture components by active molecules promoting stemness, such as KLF4 inhibitors, will open original perspectives for the development of next-generation models of skin substitutes. In this context, the use of PAK1-ROCK-Myosin II and TGF-β signaling inhibitors has been proposed to promote the preservation of stemness in expansion cultures of different types of epithelial cells [ 28 ]. Clues will certainly emerge from the combination of these converging studies ( Figure 3 ).…”

Establishment of primary transgenic human airway epithelial cell cultures to study respiratory virus – host interactions

“…This fundamental change in our primary airway epithelial cell culture protocol has been pivotal to successful cellular expansion post-lentiviral transduction and, subsequently, the establishment of transgenic hAEC cultures suitable for virus – host interaction studies. During the development and validation of our protocol other research groups have reported alternatives or improvements to the use of the Rho-kinase associated inhibitor to further extend the life-span of primary human bronchial cells [38–41]. We anticipate that the incorporation of these alternative inhibitors and methods will further improve the currently established protocol to generate homogenously transgenic hAEC cultures, and might even facilitate the adaptation of this protocol to primary airway epithelial cells from other species to further elucidate molecular virus – host interactions without using animal models.…”

Air‐liquid interface cell culture: From airway epithelium to the female reproductive tract

“…Recently, conditional reprogramming became possible also in feeder‐free, chemically defined media. Conditionally reprogrammed cells retain genome stability and differentiate under ALI conditions to fully functional epithelial tissues (Liu et al., ; Suprynowicz et al., ; Zhang et al., ).…”

“…Recently, conditional reprogramming became possible also in feeder-free, chemically defined media. Conditionally reprogrammed cells retain genome stability and differentiate under ALI conditions to fully functional epithelial tissues Suprynowicz et al, 2012;Zhang et al, 2018). In general, there are ALI culture protocols available for all epithelia lining the lumen of the FRT (examples are given in Table 1).…”