generation-human-induced-pluripotent-stem-cell-derived-planar-hair

Generation of Human Induced Pluripotent Stem Cell-derived Planar Hair-bearing Skin Organoids Using an Air-Liquid Interface Culture System

Human induced pluripotent stem cell (hiPSC)-derived skin organoids (SOs) containing hair follicles provide a sophisticated in vitro model for ...

Leiden University Medical Center

Fibroblasts from two patients carrying a heterozygous mutation in the translation initiation codon (c.2 T > G) of the kelch-like protein 24 (KLHL24) gene were used to generate human induced pluripotent stem cells (hiPSCs), using non-integrating Sendai virus to deliver reprogramming factors. CRISPR-Cas9 editing was used for genetic correction of the mutation in the patient-hiPSCs. The top-predicted off-target sites were not altered. Patient and isogenic hiPSCs showed typical morphology, expressed pluripotency-associated markers, had the capacity for in vitro differentiation into the three germ layers and displayed a normal karyotype. These isogenic pairs will enable in vitro modelling of KLHL24-associated heart and skin conditions.

Generation and genetic repair of two human induced pluripotent cell lines from patients with Epidermolysis Bullosa simplex and dilated cardiomyopathy associated with a heterozygous mutation in the translation initiation codon of KLHL24.

Human induced pluripotent stem cell (hiPSC)-derived hair-bearing skin organoids offer exciting new possibilities for modeling diseases like epidermolysis bullosa (EB). These inherited diseases affect 1 in 30,000 people worldwide and result from perturbed expression and/or structure of components of the epidermal-dermal junction (EDJ). To establish whether hiPSC-derived skin organoids might be able to capture salient features of EB, it is thus important to characterize their EDJ. Here, we report successful generation of hair-bearing skin organoids from two hiPSC lines that exhibited fully stratified interfollicular epidermis. Using immunofluorescence and electron microscopy, we showed that basal keratinocytes in organoids adhere to laminin-332 and type IV collagen-rich basement membrane via type I hemidesmosomes and integrin β1-based adhesion complexes. Importantly, we demonstrated that EDJs in organoids are almost devoid of type VII collagen, a fibril that mediates anchorage of the epidermis to dermis. This should be considered when using skin organoids for EB modeling.

Characterization of the epidermal-dermal junction in hiPSC-derived skin organoids.

Fibroblasts from two patients carrying a distinct heterozygous mutation in the KLHL24 gene (c.1 A>G and c.2T>C) were reprogrammed to obtain hiPSC lines. Non-integrating Sendai virus and CRISPR-Cas9 editing were respectively used to deliver the reprogramming factors and repair the mutation in the patient-hiPSCs to obtain isogenic control pairs. No off-target nuclease activity was detected with the top-predicted sites. Patient and isogenic hiPSCs displayed typical morphology, expressed markers of the undifferentiated state, were able to differentiate into the three germ layers and had normal karyotypes. These isogenic pairs will expand the panel of hiPSC lines to model KLHL24-associated conditions.

Generation and genetic repair of two human induced pluripotent stem cell lines from patients with Epidermolysis Bullosa simplex associated with a heterozygous mutation in the translation initiation codon of KLHL24.

Article	Year
Generation and genetic repair of two human induced pluripotent cell lines from patients with Epidermolysis Bullosa simplex and dilated cardiomyopathy associated with a heterozygous mutation ... Stem cell research\| PubMed ID: 34688992	2021
Characterization of the epidermal-dermal junction in hiPSC-derived skin organoids. Stem cell reports\| PubMed ID: 35561682	2022
Generation and genetic repair of two human induced pluripotent stem cell lines from patients with Epidermolysis Bullosa simplex associated with a heterozygous mutation in the translation ini... Stem cell research\| PubMed ID: 39317060	2024

Veronika Ramovs

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