Laminin fragments conjugated with perlecan's growth factor-binding domain differentiate human induced pluripotent stem cells into skin-derived precursor cells.

Deriving stem cells to regenerate full-thickness human skin is important for treating skin disorders without invasive surgical procedures. Our previous protocol to differentiate human induced pluripotent stem cells (iPSCs) into skin-derived precursor cells (SKPs) as a source of dermal stem cells employs mouse fibroblasts as feeder cells and is therefore unsuitable for clinical use. Herein, we report a feeder-free method for differentiating iPSCs into SKPs by customising culture substrates. We immunohistochemically screened for laminins expressed in dermal papillae (DP) and explored the conditions for inducing the differentiation of iPSCs into SKPs on recombinant laminin E8 (LM-E8) fragments with or without conjugation to domain I of perlecan (PDI), which binds to growth factors through heparan sulphate chains. Several LM-E8 fragments, including those of LM111, 121, 332, 421, 511, and 521, supported iPSC differentiation into SKPs without PDI conjugation. However, the SKP yield was significantly enhanced on PDI-conjugated LM-E8 fragments. SKPs induced on PDI-conjugated LM111-E8 fragments retained the gene expression patterns characteristic of SKPs, as well as the ability to differentiate into adipocytes, osteocytes, and Schwann cells. Thus, PDI-conjugated LM-E8 fragments are promising agents for inducing iPSC differentiation into SKPs in clinical settings.

Induction of Skin-Derived Precursor Cells from Human Induced Pluripotent Stem Cells.

The generation of full thickness human skin from dissociated cells is an attractive approach not only for treating skin diseases, but also for treating many systemic disorders. However, it is currently not possible to obtain an unlimited number of skin dermal cells. The goal of this study was to develop a procedure to produce skin dermal stem cells from induced pluripotent stem cells (iPSCs). Skin-derived precursor cells (SKPs) were isolated as adult dermal precursors that could differentiate into both neural and mesodermal progenies and could reconstitute the dermis. Thus, we attempted to generate SKPs from iPSCs that could reconstitute the skin dermis. Human iPSCs were initially cultured with recombinant noggin and SB431542, an inhibitor of activin/nodal and TGFß signaling, to induce neural crest progenitor cells. Those cells were then treated with SKP medium that included CHIR99021, a WNT signal activator. The induction efficacy from neural crest progenitor cells to SKPs was more than 97%. No other modifiers tested were able to induce those cells. Those human iPSC-derived SKPs (hiPSC-SKPs) showed a similar gene expression signature to SKPs isolated from human skin dermis. Human iPSC-SKPs differentiated into neural and mesodermal progenies, including adipocytes, skeletogenic cell types and Schwann cells. Moreover, they could be induced to follicular type keratinization when co-cultured with human epidermal keratinocytes. We here provide a new efficient protocol to create human skin dermal stem cells from hiPSCs that could contribute to the treatment of various skin disorders.

Neprilysin is identical to skin fibroblast elastase: its role in skin aging and UV responses.

Although human skin fibroblast (HSF) elastase has been characterized as a membrane-bound metalloproteinase, little is known about its structure, amino acid sequence, and encoding gene. As there are similarities in the molecular weights and inhibitory profiles of HSF elastase and neprilysin (neutral endopeptidase 24.11 (NEP)), in this study we tested the hypothesis that they are identical using immunoprecipitation and transfection methods. An immunoprecipitation study demonstrated that HSF elastase activity co-immunoprecipitated with anti-NEP in lysates of cultured HSF. Transfection of an NEP cDNA expression vector into COS-1 cells elicited the expression of HSF elastase and NEP activities in the transfected cells. These findings strongly suggest that HSF elastase is identical to NEP, which functions mainly in neuron-associated cells to degrade neuropeptides. Analysis of the expression pattern of NEP revealed that its expression was remarkably up-regulated at the gene, protein, and enzymatic activity levels during the replicative senescence of cultured HSF. Further, the activity of NEP was markedly enhanced in a pattern similar to elastase activity during the intrinsic aging of mouse skin, in UVA-exposed HSF as well as in HSF treated with conditioned medium from UVB-exposed human keratinocytes. Analysis of the cytokine profile for the stimulation of NEP and HSF elastase activities in HSF demonstrated that among the 11 cytokines tested, IL-1α, IL-1ß, IL-6, IL-8, and GM-CSF had the potential to significantly stimulate both activities similarly, again supporting the identity of HSF elastase and NEP.

Involvement of IGF-1/IGFBP-3 signaling on the conspicuousness of facial pores.

Conspicuous facial pores are one type of serious esthetic defects for many women. We previously reported that the severity of impairment of skin architecture around facial pores correlates well with the appearance of facial pores in several ethnic groups. In our last report, we showed that serum levels of insulin-like growth factor-1 (IGF-1) correlate well with facial pore size and with the severity of impairment of epidermal architecture around facial pores. However, our results could not fully explain the implication between facial pores and IGF signaling. In this study, we conducted a histological analysis of facial skin to determine whether potential changes in IGF-1 availability occur in the skin with or without conspicuous pores. Immunohistochemical observations showed that expression of insulin-like growth factor binding protein-3 (IGFBP-3) is limited to the suprapapillary epidermis around facial pores and to basal cells of rete pegs without tips in epidermis with conspicuous pores. In contrast, in basal cells of skin without conspicuous pores, IGFBP-3 expression is very low. Ki-67 and IGF-1 receptor-positive cells are abundant in basal cells in the tips of the rete pegs in skin with typical epidermal architecture around facial pores. No obvious differences were observed in the expression of filaggrin, involucrin, K1, K6 or K17 in skin with or without conspicuous pores. However, increased expression of K16 was observed in skin with conspicuous pores suggesting hyperproliferation. These results suggest that the IGF-1/IGFBP-3 signaling pathway is involved in the formation of conspicuous facial pores due to the epidermal architecture around facial pores.

Ethnic differences in the structural properties of facial skin.

BACKGROUND: Conspicuous facial pores are one type of serious aesthetic defects for many women. However, the mechanism(s) that underlie the conspicuousness of facial pores remains unclear. We previously characterized the epidermal architecture around facial pores that correlated with the appearance of those pores. OBJECTIVES: A survey was carried out to elucidate ethnic-dependent differences in facial pore size and in epidermal architecture. METHODS: The subjects included 80 healthy women (aged 30-39: Caucasians, Asians, Hispanics and African Americans) living in Dallas in the USA. First, surface replicas were collected to compare pore sizes of cheek skin. Second, horizontal cross-sectioned images from cheek skin were obtained non-invasively from the same subjects using in vivo confocal laser scanning microscopy (CLSM) and the severity of impairment of epidermal architecture around facial pores was determined. Finally, to compare racial differences in the architecture of the interfollicular epidermis of facial cheek skin, horizontal cross-sectioned images were obtained and the numbers of dermal papillae were counted. RESULTS: Asians had the smallest pore areas compared with other racial groups. Regarding the epidermal architecture around facial pores, all ethnic groups observed in this study had similar morphological features and African Americans showed substantially more severe impairment of architecture around facial pores than any other racial group. In addition, significant differences were observed in the architecture of the interfollicular epidermis between ethnic groups. CONCLUSIONS: These results suggest that facial pore size, the epidermal architecture around facial pores and the architecture of the interfollicular epidermis differ between ethnic groups. This might affect the appearance of facial pores.

Localization of ABCA12 from Golgi apparatus to lamellar granules in human upper epidermal keratinocytes.

ABCA12 is an ATP-binding cassette transporter and is thought to act as a transmembrane lipid transporter. We reported that deleterious ABCA12 mutations cause a disturbance in lamellar granule (LG) lipid transport in the epidermal granular layer keratinocytes, resulting in harlequin ichthyosis, a severe genodermatosis. Detailed localization of ABCA12 in comparison with glucosylceramide and Golgi apparatus markers were studied in order to obtain clues to clarify the function(s) of ABCA12 in human skin. We performed double-labelling immunofluorescent staining using antibodies against ABCA12, glucosylceramide and two Golgi apparatus markers (TGN46 and GM130) in normal human skin and cultured keratinocytes. Immunogold electron microscopy for ABCA12 and glucosylceramide was studied on postembedding and cryoultrathin sections of normal human skin. Confocal laser scanning microscopy demonstrated that ABCA12 and glucosylceramide co-localized in the granular layer keratinocytes as well as in keratinocytes cultured in high Ca2+ conditions through the Golgi apparatus to the cell periphery. Postembedding immunogold electron microscopy revealed that both ABCA12 and glucosylceramide labellings were associated with the LG of the uppermost granular layer keratinocytes. Using cryoultramicrotomy, lamellar structures in the LG were more clearly observed, and ultrastructural localization of ABCA12 and glucosylceramide was better demonstrated to LG in the uppermost granular layer cells. These results indicate that ABCA12 plays an important role in lipid transport from the Golgi apparatus to LG in human granular layer keratinocytes.

Expression of the keratinocyte lipid transporter ABCA12 in developing and reconstituted human epidermis.

Serious defects in the epidermal keratinocyte lipid transporter ABCA12 are known to result in a deficient skin lipid barrier, leading to harlequin ichthyosis (HI). HI is the most severe inherited keratinizing disorder and is frequently fatal in the perinatal period. To clarify the role of ABCA12, ABCA12 expression was studied in developing human skin and HI lesions artificially reconstituted in immunodeficient mice. By immunofluorescent study, ABCA12 was expressed in the periderm of the early stage two-layered human fetal epidermis. After formation of a three-layered epidermis, ABCA12 staining was seen throughout the entire epidermis. ABCA12 mRNA expression significantly increased during human skin development and reached 62% of the expression in normal adult skin, whereas the expression rate of transglutaminase 1, loricrin, and kallikrein 7 remained low. We transplanted keratinocytes from patients with HI and succeeded in reconstituting HI skin lesions in immunodeficient mice. The reconstituted lesions showed similar changes to those of patients with HI. Our findings demonstrate that ABCA12 is highly expressed in fetal skin and suggest that ABCA12 may play an essential role under both the wet and dry conditions, including the dramatic turning point from a wet environment of the amniotic fluid to a dry environment after birth.

Compound heterozygous mutations including a de novo missense mutation in ABCA12 led to a case of harlequin ichthyosis with moderate clinical severity.

Harlequin ichthyosis (HI) is one of the most devastating genodermatoses. Recently, ABCA12 mutations were identified as the cause of HI. A newborn Japanese male demonstrated the typical features of HI. The patient was treated with oral etretinate and his general condition has been good (now aged 1.5 years). This patient with moderate clinical severity was compound heterozygous for a novel de novo missense mutation 1160G > A (S387N) in exon 10 and a maternal deletion mutation 4158_4160delTAC (T1387del) in exon 28 of ABCA12. T1387del was a deletion of a highly conserved threonine residue within the first adenosine 5' triphosphate-binding domain and is thought to seriously affect the function of the ABCA12 protein. Conversely, the residue 387 is located outside the known active sites of ABCA12 and S387N is predicted not to lead to a serious functional deficiency in ABCA12. Electron microscopy revealed abnormal lamellar granules in the granular layer cells and a moderate number of lipid vacuoles in the cornified cells. Disturbed glucosylceramide transport was confirmed in the cultured keratinocytes from the patient. No de novo mutation in ABCA12 has yet been reported either in HI or lamellar ichthyosis. The present case suggested that a de novo ABCA12 mutation might underlie HI.

Expression of RNA-binding protein Musashi in hair follicle development and hair cycle progression.

Epithelial stem cells reside in the hair follicle (HF) bulge region and possess the ability to differentiate into a variety of cutaneous epithelial cells. The evolutionarily conserved Musashi family of RNA-binding proteins is associated with maintenance and/or asymmetric cell division of neural progenitor cells, and a mammalian Musashi protein is expressed in various epithelial stem/progenitor cells, including gut, stomach, and mammary gland. Thus, we hypothesized that Musashi might be expressed in stem cells and early progenitor cells of HF epithelium. Reverse transcriptase-polymerase chain reaction and immunoblotting identified Musashi-1 (Msi-1) and Musashi-2 (Msi-2) mRNA and protein in cultured mouse keratinocytes, but only Msi-1 was identified in human keratinocytes. In mice, immunohistochemical studies showed that Msi-1 and Msi-2 were expressed in the epidermis and HFs from E14.5 until adulthood. In the early anagen phase, Msi-1 and Msi-2 were expressed in the bulge and secondary germ cells and subsequently in inner root sheath (IRS) cells, especially the middle IRS cells, during the late anagen phase. In human skin, Msi-1 was detected in fetal HF cells but not in adult HFs. These observations suggest that Musashi functions not only in the asymmetric division of early progenitor cells but also in the differentiation of IRS cells during HF development and hair cycle progression.

The effect of sunscreen on skin elastase activity induced by ultraviolet-A irradiation.

It has been reported that application of sunscreens prevents the photoaging of skin in animal models and in humans. We irradiated the dorsal skin of hairless mice with ultraviolet-A (UVA), and investigated the effects of sunscreens on skin elastase activity and on skin properties. Six-week-old female HR/ICR hairless mice were used in these experiments. After being treated with either a UVA sunscreen (also containing ultraviolet-B (UVB) sunscreen to eliminate any slight UVB in the UVA lamps; Protection Factor of UVA (PFA)=6, Sun Protection Factor (SPF)=20) or a vehicle, the dorsal skins of mice were irradiated with the UVA lamps at 22.3 J/cm(2)/d, 5 times a week. At the end of 15 weeks skin properties were evaluated and elastase activities were measured. In the vehicle control group, UVA irradiation increased the brightness and yellowing of the skin, decreased the water content of the stratum corneum, increased skin thickness, decreased skin elasticity, increased skin elastase activity, and decreased the ability of the skin to recover in a pinch test, as compared to an unirradiated group. All these differences were statistically significant. In the UVA sunscreen group, both the UVA induced skin damage and the increase in skin elastase activity were significantly inhibited, as compared to the vehicle group. However, as compared to the unirradiated group, skin elastase activity was significantly increased and immediate extensibility of skin (Ue) was significantly decreased, thereby indicating that the UVA sunscreen did not prevent photoaging to the same level as the unirradiated group. These results suggest the partial efficacy of the topical photoprotection from UVA by the sunscreen in inhibiting elastase activation, and also suggest the possibility of reducing photoaging.

Mutations in lipid transporter ABCA12 in harlequin ichthyosis and functional recovery by corrective gene transfer.

Harlequin ichthyosis (HI) is a devastating skin disorder with an unknown underlying cause. Abnormal keratinocyte lamellar granules (LGs) are a hallmark of HI skin. ABCA12 is a member of the ATP-binding cassette transporter family, and members of the ABCA subfamily are known to have closely related functions as lipid transporters. ABCA3 is involved in lipid secretion via LGs from alveolar type II cells, and missense mutations in ABCA12 have been reported to cause lamellar ichthyosis type 2, a milder form of ichthyosis. Therefore, we hypothesized that HI might be caused by mutations that lead to serious ABCA12 defects. We identify 5 distinct ABCA12 mutations, either in a compound heterozygous or homozygous state, in patients from 4 HI families. All the mutations resulted in truncation or deletion of highly conserved regions of ABCA12. Immunoelectron microscopy revealed that ABCA12 localized to LGs in normal epidermal keratinocytes. We confirmed that ABCA12 defects cause congested lipid secretion in cultured HI keratinocytes and succeeded in obtaining the recovery of LG lipid secretion after corrective gene transfer of ABCA12. We concluded that ABCA12 works as an epidermal keratinocyte lipid transporter and that defective ABCA12 results in a loss of the skin lipid barrier, leading to HI. Our findings not only allow DNA-based early prenatal diagnosis but also suggest the possibility of gene therapy for HI.