iPSC-derived melanocytes express normal melanocyte markers and produce melanin.

<p><b>(a-e)</b> Normal epidermal melanocytes (NEM) and <b>(g-k)</b> iPSC-derived cells (at day 11 of the differentiation protocol) express SOX-10, MITF-M and gp-100. Phase contrast images of normal <b>(f)</b> and iPSC-derived <b>(m)</b> cells (at day 11 of the differentiation protocol) showing typical melanocyte morphology. Image <b>(l)</b> represents gp-100 expression in iPSC-derived cells at day 25 of the differentiation protocol. <b>(n)</b> At day 17 of the differentiation protocol expression of melanocyte specific markers is greatly up-regulated in iPSC-derived cells compared to iPSCs. <b>(o)</b> NEM produce melanin as shown by Fontana-Masson staining. <b>(p)</b> & <b>(q)</b> Brightfield microscopy and Fontana-Masson staining, respectively, of iPSC-derived cells (at day 28 of the differentiation protocol) containing melanin. Scale bar = 50μm. Arrows point to melanin as determined by Fontana-Masson staining.</p

Characteristics of iPSC-derived fibroblasts.

<p>(A) Cell surface marker expression in iPSC-derived fibroblasts. FACS analysis showed that the cell surface marker profile of iPSC-derived fibroblasts was very similar to normal human fibroblasts, with over 90% expression of each marker. (B) Protein expression of iPSC-derived fibroblasts. Immunostaining demonstrated that iPSC-derived fibroblasts express fibroblast markers vimentin (VIM) and prolyl-4-hydroxylase beta (P4HB), and produced multiple types of collagen as well as normal human fibroblasts. (C) Detection of mature form of type VII collagen. Western blotting analysis detected 290kD bands in each lane, indicating that type VII collagen was synthesized from iPSC-derived fibroblasts, as well as from normal fibroblasts. The lower band of 250kD may be from either degradation or an immature form of type VII collagen, since it appears to be more abundant in the cells (lanes 3 and 4) than in the culture media (lanes 1 and 2). </p

3D skin equivalents produced from iPSC-derived fibroblasts, keratinocytes and melanocytes have normal architecture and are functional.

<p><b>a)</b> Hematoxylin and eosin, <b>b)</b> Ki67, <b>c)</b> gp-100, <b>d)</b> Fontana-Masson, <b>e)</b> loricrin, <b>f)</b> keratin-1, <b>g)</b> keratin-14. Blue (DAPI), dashed line (basement membrane). Inset c) (enlarged image showing an iPSC-derived melanocyte in the basal layer of the epidermis). Inset d) (enlarged image showing an iPSC-derived melanocyte producing melanin). <b>h & i)</b> Control and forskolin-treated iPSC-derived 3D skin equivalents, respectively, after 14 days at the air-liquid interface. <b>j)</b> Quantification of melanin increase in 3D skin equivalents in response to 40μM forskolin. Scale bar = 100μm (except in enlarged images = 10μm). *** = p<0.001.</p

iPSC-derived keratinocytes internalize melanosomes in a PAR-2-dependent manner.

<p><b>a)</b> Normal epidermal keratinocytes (NHK) and iPSC-derived keratinocytes (iPSC-Ker) internalize freshly isolated melanosomes within 24 hours via a mechanism involving PAR-2.–Melanosomes (Fontana-Masson stain of normal and iPSC-derived keratinocytes after 24 hours in culture without the addition of melanosomes), + Melanosomes (Fontana-Masson stain of normal and iPSC-derived keratinocytes after 24 hours in culture with the addition of melanosomes), + Melanosomes & STI (Fontana-Masson stain of normal and iPSC-derived keratinocytes after 24 hours in culture with the addition of melanosomes and soybean trypsin inhibitor). Scale bar = 100μm. <b>b)</b> Quantification of Fontana-Masson staining in a). * = p<0.05, ** = p<0.01, *** = p<0.001.</p

Directed differentiation of iPSCs into fibroblasts.

<p>(A) Schematic representation of the differentiation strategy for generating fibroblasts from iPSCs. Cells growing out from attached embryoid bodies made in medium supplemented with AA and TGFβ2 were repeatedly split to obtain a fibroblast-like cell population. (B) Morphology of iPSC-derived fibroblasts. After 5 passages, fibroblast-like spindle-shape cells were observed. (C) Gene expression in iPSC-derived fibroblasts. Expression of stem cell markers (OCT4 and SOX2) disappeared, and markers of fibroblasts (CD10, CD73, type I and III collagen) were increased in iPSC-derived fibroblasts compared with iPSCs. </p

Lists of proteins analyzed by pathway analysis.

<p>Lists of proteins analyzed by pathway analysis.</p

Suppression of ribosomal RNA synthesis by combined KPNA knockdown.

<p>Under starvation conditions (0.1% fetal bovine serum), siRNA-mediated knockdown of KPNA2, 1, 3, and 4 significantly suppressed ribosomal RNA synthesis analyzed by reverse transcription-quantitative polymerase chain reaction (***p<0.01). The amount of pre-ribosomal RNA was reduced by about 37% after 72 h.</p

Overexpression of KPNA2 in proliferating cells.

<p>Immunohistochemistry showed KPNA2 was uniformly expressed throughout the epidermis in healthy skin, although KPNA2 overexpression was observed in the basal layer in psoriasis. In contrast, very few cells exhibited KPNA2 staining in the basal cells of atopic dermatitis. KPNA2 overexpression was observed in the tumor cells of Bowen’s disease, actinic keratosis, squamous cell carcinoma, Paget’s disease, Merkel cell carcinoma, and mycosis fungoides.</p

Suppression of cell growth by combined KPNA knockdown.

<p>Under starvation conditions (0.1% FBS), siRNA-mediated knockdown of KPNA2, 1, 3, and 4 suppressed cell growth after 120 h (*p<0.05). Only KPNA2 siRNA subtraction produced no change in proliferation.</p

Suppression of protein synthesis by combined KPNA knockdown.

<p>Under starvation conditions (0.1% fetal bovine serum), siRNA-mediated knockdown of KPNA2, 1, 3, and 4 significantly suppressed protein synthesis after 48 h (*p<0.05) and 72 h (***p<0.01), as demonstrated by metabolic labeling with ³⁵S-methionine.</p