ABSTRACT
Atopic dermatitis (AD), driven by interleukins (IL-4/IL-13), is a chronic inflammatory skin disease characterized by intensive pruritus. However, it is unclear how immune signaling and sensory response pathways cross talk with each other. We differentiated itch sensory neuron-like cells (ISNLCs) from iPSC lines. These ISNLCs displayed neural markers and action potentials and responded specifically to itch-specific stimuli. These ISNLCs expressed receptors specific for IL-4/IL-13 and were activated directly by the two cytokines. We successfully innervated these ISNLCs into full thickness human skin constructs. These innervated skin grafts can be used in clinical applications such as wound healing. Moreover, the availability of such innervated skin models will be valuable to develop drugs to treat skin diseases such as AD.
Subject(s)
Carcinoma, Squamous Cell/drug therapy , Epidermolysis Bullosa Dystrophica/drug therapy , Janus Kinases/antagonists & inhibitors , Pyrazoles/administration & dosage , Skin Neoplasms/drug therapy , Administration, Oral , Animals , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/pathology , Cell Line, Tumor , Collagen Type VII/genetics , Epidermolysis Bullosa Dystrophica/complications , Epidermolysis Bullosa Dystrophica/genetics , Fibroblasts , Humans , Janus Kinases/metabolism , Mice , Mutation , Nitriles , Pyrazoles/pharmacology , Pyrimidines , STAT3 Transcription Factor/metabolism , Signal Transduction/drug effects , Skin Neoplasms/genetics , Skin Neoplasms/pathology , Xenograft Model Antitumor AssaysABSTRACT
Recessive dystrophic epidermolysis bullosa (RDEB) is a severe inherited skin disorder caused by mutations in the COL7A1 gene encoding type VII collagen (C7). The spectrum of severity depends on the type of mutation in the COL7A1 gene. C7 is the major constituent of anchoring fibrils (AFs) at the basement membrane zone (BMZ). Patients with RDEB lack functional C7 and have severely impaired dermal-epidermal stability, resulting in extensive blistering and open wounds on the skin that greatly affect the patient's quality of life. There are currently no therapies approved for the treatment of RDEB. Here, we demonstrated the correction of mutations in exon 19 (c.2470insG) and exon 32 (c.3948insT) in the COL7A1 gene through homology-directed repair (HDR). We used the clustered regulatory interspaced short palindromic repeats (CRISPR) Cas9-gRNAs system to modify induced pluripotent stem cells (iPSCs) derived from patients with RDEB in both the heterozygous and homozygous states. Three-dimensional human skin equivalents (HSEs) were generated from gene-corrected iPSCs, differentiated into keratinocytes (KCs) and fibroblasts (FBs), and grafted onto immunodeficient mice, which showed normal expression of C7 at the BMZ as well as restored AFs 2 mo postgrafting. Safety assessment for potential off-target Cas9 cleavage activity did not reveal any unintended nuclease activity. Our findings represent a crucial advance for clinical applications of innovative autologous stem cell-based therapies for RDEB.
