Y‐27632 simplifies the isolation procedure of human primary epidermal cells by selectively blocking focal adhesion of dermal cells

Abstract

Primary skin epidermal cells isolation and in vitro expansion culture have been widely used for laboratory research and clinical applications. The conventional methods involving sequential enzymatic digestion of adult tissues have given low cell recovery rate and reduced cell viability. We report here an advanced method for human primary epidermal progenitor cells isolation from skin tissues including the Rho kinase inhibitor Y-27632. Compared with traditional protocols, the current protocol is simple, easy, and faster; moreover, it gives a greater yield of integrin-expressing epithelial stem cells. In addition, our new methodology does not require a separation of epidermis from dermis because the medium selectively blocks focal adhesion and growth of dermal cells. Importantly, the cells isolated from this method can maintain their regeneration potential and quickly reconstitute a mature human skin in vivo after grafting onto nude mice. In brief, we describe here a simple (one step) and serum-free method for isolating primary epidermal stem cells from adult tissues. The isolated cells may be widely used for both laboratory studies and clinical application, especially in the field of tissue engineering and regeneration.

The crosstalk between breast carcinoma-associated fibroblasts and cancer cells promotes RhoA-dependent invasion via IGF-1 and PAI-1

“…ROCK inhibition is known to facilitate the growth and survival of human embryonic stem cells by inhibiting anoikis. ROCK inhibitors are now part of standard stem cell culture protocols [ 51 , 52 ]. Our results show that Y27632 can also increase the in vitro viability of MDA-MB-231 spheroids ( Supplementary Figure 8 ).…”

Prolonged treatment with Y-27632 promotes the senescence of primary human dermal fibroblasts by increasing the expression of IGFBP-5 and transforming them into a CAF-like phenotype

“…Y-27632 has been reported to promote the proliferation and migration of keratinocytes [ 23 , 31 ]. Interestingly, when we cultured a mixture of epidermal cells and dermal cells from the skin in the presence of Y-27632, we found that the growth of epidermal cells was significantly increased but the growth of dermal cells was clearly inhibited [ 32 ], which was inconsistent with a report by Piltti et al showing that Y-27632 increased the proliferation of dermal fibroblasts [ 32 , 33 ]. Therefore, the detailed role of Y-27632 on the growth of skin dermal fibroblasts needs to be further clarified.…”

The ARE-binding protein Tristetraprolin (TTP) is a novel target and mediator of calcineurin tumor suppressing function in the skin

“…In order to obtain CnB1 KO MKCs, MKCs from CnB1 flox/flox transgenic mice (see below) were infected with an adenovirus expressing Cre (Ad-Cre) or GFP (Ad-GFP, as a control). Primary HKCs were isolated from foreskin tissues and were cultured in serum-free keratinocyte medium (SFM, Invitrogen) as previously described [ 39 – 41 ]. All SCC lines (SCC12, 13, 15, 25 were derived from the skin, and SCCO11, O12, O22, O23, O28 were derived from the oral epithelium) were cultured in SFM.…”