Prolonging culture of primary human keratinocytes isolated from suction blisters with the Rho kinase inhibitor Y-27632

Abstract

Keratinocytes are the most abundant cell type in the epidermis. They prevent desiccation and provide immunological and barrier defense against potential pathogens such as Staphylococcus aureus and Candida albicans. The study of this first line of immune defense may be hindered by invasive isolation methods and/or improper culture conditions to support stem cell maintenance and other potential mechanisms contributing to long-term subcultivation in vitro. Primary keratinocytes have been successfully isolated from blister roofs induced by negative pressure, which separates the epidermis from the dermis in vivo in human subjects. This method allows collection of pure epidermal cells without dermal contamination in a minimally invasive manner. However, the isolated keratinocytes differentiate and senesce when cultured in vitro beyond five passages. Here, we present evidence that the Rho kinase (ROCK) inhibitor Y-27632 can be used to effectively increase the proliferative capabilities of keratinocytes isolated using the suction blister method, similar to what has been previously reported for primary keratinocytes isolated using alternative methods. We show that the increase in passage number is directly correlated to delayed differentiation, and that cells passaged long term with the inhibitor retain their ability to stratify in organotypic raft cultures and respond to cytokine treatment; additionally, the late passage cells have a heterogeneous mix of differentiated and non-differentiated cells which may be predicted by a ratio of select differentiation markers. The described method presents a minimally invasive procedure for keratinocyte isolation and prolonged culture that allows analysis of keratinocyte function in both healthy volunteers and patients with dermatologic diseases.

RAS induced senescence of skin keratinocytes is mediated through Rho‐associated protein kinase (ROCK)

“…Pharmacological ROCK inhibitors were reported to block keratinocyte terminal differentiation. [7][8][9] Paradoxically, knockdown of ROCK by siRNA in primary mouse keratinocytes enhanced expression of two early markers of keratinocyte differentiation, keratin 1 (K1) and 10 (K10) (Figure 1A), independent of extracellular calcium, a known regulator of these markers. 21 Overexpression of wildtype ROCK, but not a kinase-dead (KD) mutant, suppressed the expression of K1 and K10 (Figure 1B).…”

“…Several small-molecule inhibitors of the Rho-associated protein kinase (ROCK) have been shown to protect specific normal and malignant cell types from anoikis, enhance viability in both culture and cryopreservation, and prolong long-term survival. [6][7][8] This discovery was particularly useful to preserve both mouse and human keratinocytes that otherwise differentiate and senesce in vitro. 8,9 However, it has not been demonstrated whether ROCK might mediate oncogene-induced senescence as a consequence of RAS transformation of keratinocytes.…”

Therapeutic candidates for keloid scars identified by qualitative review of scratch assay research for wound healing

“…Cells form keloid scars are known to display Warburg metabolism-a form of metabolic disruption associated with cancer cells in which over expression of STAT3, in conjunction with JAK2, drives a tendency for rapidly proliferating cells to generate ATP via glycolysis rather than OxPhos, even with available oxygen [222,223]. Consistent with these reports, keloid…”

hiPSC-Derived Epidermal Keratinocytes from Ichthyosis Patients Show Altered Expression of Cornification Markers

“…We show here that treatment with Rho kinase inhibitor Y-27632 significantly improves the morphology and proliferation ability of hiPSC-bKs. Accordingly, this compound has been shown to improve survival and proliferation of both primary keratinocytes as well as hiPSC-derived retinal pigmented cells [ 35 , 36 ]. Importantly, Y-27632 might have inhibitory effects on keratinocyte terminal differentiation [ 37 ]; therefore, it was only added during hiPSC-bK expansion.…”

“…Importantly, Y-27632 might have inhibitory effects on keratinocyte terminal differentiation [ 37 ]; therefore, it was only added during hiPSC-bK expansion. Senescence of primary keratinocytes treated with Y-27632 was reported after ~70 days in culture [ 35 ]; Y-27632 supplementation does not represent a source of immortalized keratinocytes.…”