Fig 2: Keratinocyte derivation procedure in defined conditions. See also Figure S4. A. Schematic drawing of the optimized keratinocyte differentiation protocol. B. Cell morphology changes in the differentiation process. Scale bar,100µm. C. Stagewise keratinocyte gene expression by RT-qPCR. The results were normalized to hESC (D0); Data are representative of 3 independent experiments. D. Immunostaining of TP63 (red), KRT14 (green), KRT1 (green) and KRT10 (green) on day 24 and day 32 of differentiation. Scale bar, 50µm. E. Flow cytometry analysis of KRT14 (left panel) and KRT1 (right panel) on Day 28 of differentiation. Blue peak represents undifferentiated hESCs; pink peak represents keratinocyte. F. Keratinocyte differentiation from multiple hESC (H1, H9) and hiPSC (ND1-4, NL-1, NL-4) lines. Gene expression was analyzed on day 20 of differentiation and compared with HaCaT keratinocyte cell line, primary human foreskin keratinocytes (HFK-1, HFK-2) and undifferentiated hESCs (H1). The results were normalized to hESC (H1); Data are representative of 3 independent experiments.

Fig 3: Further analysis of derived keratinocytes by global gene expression and transplantation. A. Cluster of global gene expression profiles during keratinocyte differentiation. RNA samples are collected at each stage of keratinocyte differentiation, and gene expression was analyzed by microarray. The global gene expression is clustered by Complete-linkage clustering method. B. Expression of epidermal-specific genes through the differentiation process. Epidermal specific genes were selected according to published dataset, and the pattern of gene expression was displayed by stage of differentiation. C. hESC-derived keratinocyte (hKC) transplantation in mouse excisional wound model. a. Representative image of mouse excisional wound and sites of hKC application. b. Representative image of the wound right after transplantation. Exposed wound areas were marked with yellow dash line. c. Representative image of the wound seven days after wounding and cell transplantation. The skin area was processed for further study. d. H&E staining of the wound healing site seven days after keratinocyte transplantation. Square marks the area shown as high magnification image on the right. Scale bar, 400µm on the left; 100µm on the right. D. Immunostaining of KRT14, KRT1, KRT10, IVL and FLG (red fluorescence) on serial skin sections. Samples were collected seven days after transplantation of keratinocytes derived from GFP-labeled hESCs (green fluorescence). Blue fluorescence, nuclear staining with Hoechst. Scale bar, 100µm on the left; 30µm on the right. E. The percentage of GFP-positive cells expressing specific maturation marker genes, representing keratinocyte maturation in vivo. n=6. Data are presented as the mean±SD of three independent experiments.

Fig 4: Expression of Krt10, Krt1 and Krt14 in MGs of the two young donors, one with normal-appearing MGs (36F) and the other with evidence of ductal obstruction (30M). (A, B) Krt10 was expressed in the suprabasal layers of ductules and central duct in 36F (A, red arrows). In the central duct of 30M, the Krt10-positive suprabasal layers (inset in B, red arrow) was ‘sandwiched’ between two Krt10-negative layers, presumably the basal layer (black arrow) and the superficial layer (open arrow). Ductal blockage (arrowhead in B) was caused by Krt10-negative proteins mixed with cell nuclear fragments. (C, D) Krt1 expression level in the MGs of 36F and 30M was high in the ductules and reduced in the suprabasal layer of the central duct (as indicated by arrows in C). Similar to the Krt10 pattern seen in MGs of 30M (B), the superficial layer of the central duct in this donor was negative for Krt1 (open arrows in D). (E, F) Krt14 was highly expressed in the MG acinar and ductule epithelia in both 36F (E) and 30M (F). Krt14 expression was limited to the basal epithelium in the central duct in 36F (arrows in E). The thickness of the Krt14-positive layer was notably increased in the central duct of 30M (F, black arrow in the inset). Scale bar denotes 100 µm. 36F, 36-year-old female; 30M, 30-year-old male; a, acinus; Cd, central duct; d, ductule; MG, meibomian gland.

Fig 5: Details of the KRT1:c.567_569del, p.(Asn190del) variant.(A) Integrative Genomics Viewer (IGV) screenshot showing the short-read alignments of the ichthyosis affected puppy and its non-affected parents at the position of the deletion. A deletion of one copy of the allele is visible in the case but not in the parents. Note that in the IGV screenshot bases 2,422,713–2,422,715 are deleted, whereas the 3’-rule of HGVS nomenclature requires to designate this variant as Chr27:2,422,716_2,422,718del (CanFam3.1). (B) Schematic representation of the protein domain structure of a keratin dimer [20] with the highly conserved amino acid sequence of the coil 1A subdomain shown below. The variant is predicted to delete an asparagine residue from coil 1A, which is located within the rod domain of KRT1.