Short-Term Treatment with Rho-Associated Kinase Inhibitor Preserves Keratinocyte Stem Cell Characteristics In Vitro

Abstract

Primary keratinocytes including keratinocyte stem cells (KSCs) can be cultured as epidermal sheets in vitro and are attractive for cell and gene therapies for genetic skin disorders. However, the initial slow growth of freshly isolated keratinocytes hinders clinical applications. Rho-associated kinase inhibitor (ROCKi) has been used to overcome this obstacle, but its influence on the characteristics of KSC and its safety for clinical application remains unknown. In this study, primary keratinocytes were treated with ROCKi Y-27632 for six days (short-term). Significant increases in colony formation and cell proliferation during the six-day ROCKi treatment were observed and confirmed by related protein markers and single-cell transcriptomic analysis. In addition, short-term ROCKi-treated cells maintained their differentiation ability as examined by 3D-organotypic culture. However, these changes could be reversed and became indistinguishable between treated and untreated cells once ROCKi treatment was withdrawn. Further, the short-term ROCKi treatment did not reduce the number of KSCs. In addition, AKT and ERK pathways were rapidly activated upon ROCKi treatment. In conclusion, short-term ROCKi treatment can transiently and reversibly accelerate initial primary keratinocyte expansion while preserving the holoclone-forming cell population (KSCs), providing a safe avenue for clinical applications.

Dawnn: single-cell differential abundance with neural networks

“…On the keratinocyte dataset [9], we again found that Dawnn and CNA identified a larger proportion of DA regions than Milo and DA-seq (Figure 3b). Dawnn had higher TPRs than CNA for larger simulated log-fold changes.…”

“…We follow the benchmarking of Dann et al (Section 4.2.2), creating simulated and real datasets using their described approach [1]. We further assessed real-world performance using three additional biological datasets: one from a publication of ours examining the effects of accelerating skin sheet growth [9]; another from a publication characterising organoids of bile ducts [10]; and a large dataset comprising cells from the heart in a study of congenital heart disease [11]. Table 1 summarises our benchmarking datasets.…”

Bio‐electrospraying 3‐D Organotypic Human Skin Cultures

“…However, it is important to note that Jayarajan et al, used de‐epidermalized dermis as a base for generating such matured cultures in vitro. [ 11 ] Rather, the main focus of this study was to elucidate the compatibility and feasibility of bio‐electrospraying human primary fibroblasts in combination with a collagen hydrogel and subsequently keratinocytes in complete media, but not on de‐epidermalized dermis.…”

“…HKs were cultured as previously described. [ [ 11 Briefly, HKs were cultivated in a humidified atmosphere at 37 °C with 10% CO 2 in Green's medium containing DMEM and DMEM/F12 in the ratio of 1:1 supplemented with 10% FCS (Labtech, Heathfield, UK, # FCS‐SA), 50 U mL −1 penicillin and streptomycin (Gibco, Paisley, UK, # 15 070 063), 10 ng mL −1 EGF (Peprotech, London, UK, # AF‐100‐15‐1000), 0.4 µg mL −1 Hydrocortisone (Sigma, Gillingham, UK, # H‐2270), 5 µg mL −1 Transferrin (Sigma, # T2252), 5 µg mL −1 Insulin (Sigma, # I‐5500), 2 × 10 −11 m Liothyronine sodium salt (Sigma, # T6397), and 1 × 10 −10 m Cholera toxin (Sigma, # C‐8052). keratinocytes were passaged and reseeded at a density of 1.5 × 10 4 cm −2 .…”

Decoding the Human Epidermal Complexity at Single-Cell Resolution

“…For each location, references to the papers that performed the biopsies are listed in chronological order. (A) Foreskin [31][32][33]; (B) Scalp [31,34,35]; (C) Body trunk [25,31,34,36]; (D) foot [37,38]; (E) hip [38]; (F) hand [34,38]; (G) inguinal region [37,39]; (H) leg [40]; (I) buttocks [41,42]; (J) arm [30,34,40]; (K) face (forehead, eyelid, and tragus) [34,[43][44][45]; (L) neck [43]. to be analyzed with single-cell techniques.…”