FIGURE 5.

Patient-derived cells can serve as promising research tools for studying rare human diseases. Our cohort of tissue donors encompasses individuals affected by (A) Van der Woude syndrome (VWS), (B) Patau syndrome, and (C) Goldenhar syndrome (GH). Overview: A brief description of the rare human diseases is shown as well as the clinical appearance of the tissue donors. Arrowheads indicate typical phenotypes such as bilateral lip pits and orofacial clefts (VWS1, A), and pathological skin tags in the GH-affected individual (C). Biopsies: High magnification images of the tissue biopsies taken for histological analysis are depicted. Note that the biopsies shown for the VWS were all derived from a secondary surgery of a 19 years old individual (VWS2) and are indicated by numbers (1, 3, 4) in the “Overview” (A). Patient-derived cells: Explant cultures allowed the isolation of patient-derived cells of the biopsies shown, which were thoroughly characterized by morphological as well as immunofluorescent analyses. All keratinocytes (orange) show the typical colony-forming morphology (Live Imaging) and are all positive for the epithelial marker E-Cadherin (E-Cad), while fibroblasts (green) present as elongated cells (Live Imaging) positive for the mesenchymal marker Fibronectin (FN). Note that explant cultures from the scar and the “lip pits” of the VWS2 individual (A) only resulted in fibroblasts. Scale bars: 25 μm (Live Imaging keratinocytes), 50 μm (Live Imaging fibroblasts), 25 μm (IF). Histology: Representative Toluidine Blue and/or H&E stainings of the tissues, with some close-ups are shown. For the VWS2-derived samples additional immunohistochemical analyses (IHC) were performed (A). The expression of E-Cad indicating the skin epithelium was revealed in the lower lip sample. Similarly, the scar tissue revealed excessive staining for α-smooth muscle actin (α-SMA). Note also the abundant amount of striated muscles in the biopsy derived from the VWS2 “lip pits” connective tissue [anti-myosin II (MYOII) immunoreactivity and H&E/Toluidine Blue staining]. In the H&E staining of the Patau syndrome biopsy (B) the transition zone between skin and mucosa of the lip can be appreciated (Vermillion border). Finally, Safranin-O staining of the skin tag biopsied from the GH patient identifies the presence of pathological cartilage formation (C). Scale bar: 0.1 mm. In vitro characteristics: (A) qPCR and immunoblot analyses show that the levels of IRF6 in VWS1 and VWS2 keratinocytes are significantly decreased compared to the levels in 4 non-syndromic [wild-type for the VWS-associated genes IRF6 (NM_006147.4), GRHL3 (NM_198173), and NME1 (NM_198175)] CLP and one healthy lip keratinocyte cell cultures. These results strongly suggest that the VWS2 individual harbors an IRF6 variant [this has been confirmed for VWS1 by genetic analysis (Degen et al., 2020)] (left). Additionally, fibroblasts isolated from the scar tissue of VWS2 showed higher expression of α-SMA and Collagen type III (COL3) mRNA levels compared to fibroblasts isolated from the same donor but from different origins (gingiva, lower lip, and “lip pits,” right). Thus, the original characteristics are retained in the patient-derived cells. Data are expressed as mean ± SD. n = 3. * = p < 0.05. (B) Presence of three chromosomes 13 in the patient affected by the Patau syndrome is shown by karyotype analysis (red box).