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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

University of Grenoble Alpes

Hereditary Hemorrhagic Telangiectasia type 2 (HHT2) is an inherited genetic disorder characterized by vascular malformations and hemorrhage. HHT2 results from ACVRL1 haploinsufficiency, the remaining wild-type allele being unable to contribute sufficient protein to sustain endothelial cell function. Blood vessels function normally but are prone to respond to angiogenic stimuli, leading to the development of telangiectasic lesions that can bleed. How ACVRL1 haploinsufficiency leads to pathological angiogenesis is unknown.

Decreased Expression of Vascular Endothelial Growth Factor Receptor 1 Contributes to the Pathogenesis of Hereditary Hemorrhagic Telangiectasia Type 2.

Lymphoblast cells from four individuals of a family of two genetically unrelated parents and their monozygotic twins were used to generate integration-free human induced pluripotent stem cells (hiPSCs). Reprogramming factors were delivered by co-electroporation of three episomal-based plasmids expressing OCT3/4, SOX2, KLF4, L-MYC and LIN28. The hiPSCs showed a normal karyotype, expressed pluripotency-associated markers, displayed the capacity for in vitro differentiation into the three germ layers and were Epstein Barr virus-free. These hiPSC lines offer the possibility to compare genetically unrelated and genetically identical tissues from different individuals and to study genotype-specific effects, which are particularly relevant for toxicology testing.

Lymphoblast-derived hiPS cell lines generated from four individuals of a family of genetically unrelated parents and their female monozygotic twins.

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.

Mpox virus (MPXV) primarily infects human skin to cause lesions. Currently, robust models that recapitulate skin infection by MPXV are lacking. Here we demonstrate that human induced pluripotent stem cell-derived skin organoids are susceptible to MPXV infection and support infectious virus production. Keratinocytes, the predominant cell type of the skin epithelium, effectively support MPXV infection. Using transmission electron microscopy, we visualized the four stages of intracellular virus particle assembly: crescent formation, immature virions, mature virions and wrapped virions. Transcriptional analysis showed that MPXV infection rewires the host transcriptome and triggers abundant expression of viral transcripts. Early treatment with the antiviral drug tecovirimat effectively inhibits infectious virus production and prevents host transcriptome rewiring. Delayed treatment with tecovirimat also inhibits infectious MPXV particle production, albeit to a lesser extent. This study establishes human skin organoids as a robust experimental model for studying MPXV infection, mapping virus-host interactions and testing therapeutics.

Mpox virus infection and drug treatment modelled in human skin organoids.

Few human induced pluripotent stem cell (hiPSC) lines are Good Manufacturing Practice (GMP)-compliant, limiting the clinical use of hiPSC-derived products. Here, we addressed this by establishing and validating an in-house platform to produce GMP-compliant hiPSCs that would be appropriate for producing both allogeneic and autologous hiPSC-derived products.

Good Manufacturing Practice-compliant human induced pluripotent stem cells: from bench to putative clinical products.

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.

Xeroderma pigmentosum group A (XPA) is an inherited skin disorder characterized by sensitivity to ultraviolet radiation. In Maghrebi patients, a homozygous mutation in exon 6 of the XPA gene (c.682C>T) results in the introduction of a premature termination codon. Using CRISPR/Cas9-mediated gene editing, this mutation was introduced into the well-characterized LUMCi004-A line. The resulting hiPSC line showed typical morphology, expressed markers of the undifferentiated state, was able to differentiate into the three germ layers in vitro and displayed a normal karyotype. When paired with its isogenic counterpart, this line represents a valuable resource to model the disease.

Generation of XPA p.Arg228T mutant LUMCi004-A cell line for modeling Xeroderma pigmentosum group A.

Chronic wounds represent a major therapeutic challenge, with limited effective treatment options currently available. Both cellular and acellular approaches are being explored to address this issue, with mesenchymal stromal cells (MSCs) emerging as a promising option. While these cells have been extensively studied, alternative stromal cell sources, such as fibroblasts (Fbs), may also possess comparable therapeutic potential. Thus, this review focuses on stromal cell-derived secretomes (conditioned medium) as a source of acellular therapy for chronic wounds and presents the available wound-healing models (in vitro, ex vivo, and in vivo) suitable for evaluating their therapeutic efficacy, prior to clinical application. By conducting an analysis of the existing studies, we present the impact of the cell culture conditions on the enhancement in the bioactivity of the MSC/Fb-derived conditioned medium, a research area that continues to evolve.

Article	Year
Decreased Expression of Vascular Endothelial Growth Factor Receptor 1 Contributes to the Pathogenesis of Hereditary Hemorrhagic Telangiectasia Type 2. Circulation\| PubMed ID: 30571259	2018
Lymphoblast-derived hiPS cell lines generated from four individuals of a family of genetically unrelated parents and their female monozygotic twins. Stem cell research\| PubMed ID: 31734644	2019
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
Mpox virus infection and drug treatment modelled in human skin organoids. Nature microbiology\| PubMed ID: 37828248	2023
Good Manufacturing Practice-compliant human induced pluripotent stem cells: from bench to putative clinical products. Cytotherapy\| PubMed ID: 38483359	2024
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
Generation of XPA p.Arg228T mutant LUMCi004-A cell line for modeling Xeroderma pigmentosum group A. Stem cell research\| PubMed ID: 39321748	2024
Innovative Strategies: Use of Stromal Cell-Derived Secretome for Chronic Wound Therapy. International journal of molecular sciences\| PubMed ID: 40565072	2025

Karine Raymond

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