Vitiligo non-responding lesions to narrow band UVB have intriguing cellular and molecular abnormalities that may prevent epidermal repigmentation.

We have discovered that human vitiligo patients treated with narrow-band UVB (NBUVB) demonstrated localized resistance to repigmentation in skin sites characterized by distinct cellular and molecular pathways. Using immunostaining studies, discovery-stage RNA-Seq analysis, and confirmatory in situ hybridization, we analyzed paired biopsies collected from vitiligo lesions that did not repigment after 6 months of NBUVB treatment (non-responding) and compared them with repigmented (responding) lesions from the same patient. Non-responding lesions exhibited acanthotic epidermis, had low number of total, proliferative, and differentiated melanocyte (MC) populations, and increased number of senescent keratinocytes (KCs) and of cytotoxic CD8+ T cells as compared with responding lesions. The abnormal response in the non-responding lesions was driven by a dysregulated cAMP pathway and of upstream activator PDE4B, and of WNT/ß-catenin repigmentation pathway. Vitiligo-responding lesions expressed high levels of WNT10B ligand, a molecule that may prevent epidermal senescence induced by NBUVB, and that in cultured melanoblasts prevented the pro-melanogenic effect of α-MSH. Understanding the pathways that govern lack of NBUVB-induced vitiligo repigmentation has a great promise in guiding the development of new therapeutic strategies for vitiligo.

The Development of an Advanced Model for Multilayer Human Skin Reconstruction In Vivo.

Human skin reconstruction on immune-deficient mice has become indispensable for in vivo studies performed in basic research and translational laboratories. Further advancements in making sustainable, prolonged skin equivalents to study new therapeutic interventions rely on reproducible models utilizing patient-derived cells and natural three-dimensional culture conditions mimicking the structure of living skin. Here, we present a novel step-by-step protocol for grafting human skin cells onto immunocompromised mice that requires low starting cell numbers, which is essential when primary patient cells are limited for modeling skin conditions. The core elements of our method are the sequential transplantation of fibroblasts followed by keratinocytes seeded into a fibrin-based hydrogel in a silicone chamber. We optimized the fibrin gel formulation, timing for gel polymerization in vivo, cell culture conditions, and seeding density to make a robust and efficient grafting protocol. Using this approach, we can successfully engraft as few as 1.0 × 106 fresh and 2.0 × 106 frozen-then-thawed keratinocytes per 1.4 cm2 of the wound area. Additionally, it was concluded that a successful layer-by-layer engraftment of skin cells in vivo could be obtained without labor-intensive and costly methodologies such as bioprinting or engineering complex skin equivalents. Key features • Expands upon the conventional skin chamber assay method (Wang et al., 2000) to generate high-quality skin grafts using a minimal number of cultured skin cells. • The proposed approach allows the use of frozen-then-thawed keratinocytes and fibroblasts in surgical procedures. • This system holds promise for evaluating the functionality of skin cells derived from induced pluripotent stem cells and replicating various skin phenotypes. • The entire process, from thawing skin cells to establishing the graft, requires 54 days. Graphical overview.

A scalable, GMP-compatible, autologous organotypic cell therapy for Dystrophic Epidermolysis Bullosa.

Background: Gene editing in induced pluripotent stem (iPS) cells has been hailed to enable new cell therapies for various monogenetic diseases including dystrophic epidermolysis bullosa (DEB). However, manufacturing, efficacy and safety roadblocks have limited the development of genetically corrected, autologous iPS cell-based therapies. Methods: We developed Dystrophic Epidermolysis Bullosa Cell Therapy (DEBCT), a new generation GMP-compatible (cGMP), reproducible, and scalable platform to produce autologous clinical-grade iPS cell-derived organotypic induced skin composite (iSC) grafts to treat incurable wounds of patients lacking type VII collagen (C7). DEBCT uses a combined high-efficiency reprogramming and CRISPR-based genetic correction single step to generate genome scar-free, COL7A1 corrected clonal iPS cells from primary patient fibroblasts. Validated iPS cells are converted into epidermal, dermal and melanocyte progenitors with a novel 2D organoid differentiation protocol, followed by CD49f enrichment and expansion to minimize maturation heterogeneity. iSC product characterization by single cell transcriptomics was followed by mouse xenografting for disease correcting activity at 1 month and toxicology analysis at 1-6 months. Culture-acquired mutations, potential CRISPR-off targets, and cancer-driver variants were evaluated by targeted and whole genome sequencing. Findings: iPS cell-derived iSC grafts were reproducibly generated from four recessive DEB patients with different pathogenic mutations. Organotypic iSC grafts onto immune-compromised mice developed into stable stratified skin with functional C7 restoration. Single cell transcriptomic characterization of iSCs revealed prominent holoclone stem cell signatures in keratinocytes and the recently described Gibbin-dependent signature in dermal fibroblasts. The latter correlated with enhanced graftability. Multiple orthogonal sequencing and subsequent computational approaches identified random and non-oncogenic mutations introduced by the manufacturing process. Toxicology revealed no detectable tumors after 3-6 months in DEBCT-treated mice. Interpretation: DEBCT successfully overcomes previous roadblocks and represents a robust, scalable, and safe cGMP manufacturing platform for production of a CRISPR-corrected autologous organotypic skin graft to heal DEB patient wounds.

Studying Immunotherapy Resistance in a Melanoma Autologous Humanized Mouse Xenograft.

Resistance to immunotherapy is a significant challenge, and the scarcity of human models hinders the identification of the underlying mechanisms. To address this limitation, we constructed an autologous humanized mouse (aHM) model with hematopoietic stem and progenitor cells (HSPC) and tumors from 2 melanoma patients progressing to immunotherapy. Unlike mismatched humanized mouse (mHM) models, generated from cord blood-derived HSPCs and tumors from different donors, the aHM recapitulates a patient-specific tumor microenvironment (TME). When patient tumors were implanted on aHM, mHM, and NOD/SCID/IL2rg-/- (NSG) cohorts, tumors appeared earlier and grew faster on NSG and mHM cohorts. We observed that immune cells differentiating in the aHM were relatively more capable of circulating peripherally, invading into tumors and interacting with the TME. A heterologous, human leukocyte antigen (HLA-A) matched cohort also yielded slower growing tumors than non-HLA-matched mHM, indicating that a less permissive immune environment inhibits tumor progression. When the aHM, mHM, and NSG cohorts were treated with immunotherapies mirroring what the originating patients received, tumor growth in the aHM accelerated, similar to the progression observed in the patients. This rapid growth was associated with decreased immune cell infiltration, reduced interferon gamma (IFNγ)-related gene expression, and a reduction in STAT3 phosphorylation, events that were replicated in vitro using tumor-derived cell lines. IMPLICATIONS: Engrafted adult HSPCs give rise to more tumor infiltrative immune cells, increased HLA matching leads to slower tumor initiation and growth, and continuing immunotherapy past progression can paradoxically lead to increased growth.

Tracing the Equilibrium Phase of Cancer Immunoediting in Epidermal Neoplasms via Longitudinal Intravital Imaging.

Recognition of transformed cells by the immune system can sometimes generate a rate-limiting equilibrium phase, wherein tumor outgrowth is prevented without complete neoplasm elimination. Targeting premalignancies during this immune-controlled bottleneck is a promising strategy for rational cancer prevention. Thus far, immune equilibrium has been difficult to model in a traceable way, and most immunoediting systems have been limited to mesenchymal tumor types. Here, we introduce a mouse model for fluorescent tracing of somatic epithelial transformation. We demonstrate that transplantation can be used to prevent a confounding artificial tolerance that affects autochthonous inducible models. Using this system, we observe the expected dichotomy of outcomes: immune-deficient contexts permit rapid tumorigenesis, whereas initiated clones in immunocompetent mice undergo elimination or equilibrium. The equilibrium phase correlates with localization within hair follicles, which have been characterized previously as relatively immune-privileged sites. Given this, we posit that valleys in the immune surveillance landscape of a normal tissue can provide a cell-extrinsic alternative to the canonical cell-intrinsic adaptations believed to establish the equilibrium phase. Our model is a prototype for tracing immunoediting in vivo and could serve as a novel screening platform for therapies targeted against immune-controlled premalignancies.

Repigmentation of Human Vitiligo Skin by NBUVB Is Controlled by Transcription of GLI1 and Activation of the ß-Catenin Pathway in the Hair Follicle Bulge Stem Cells.

Vitiligo repigmentation is a complex process in which the melanocyte-depleted interfollicular epidermis is repopulated by melanocyte precursors from hair follicle bulge that proliferate, migrate, and differentiate into mature melanocytes on their way to the epidermis. The strongest stimulus for vitiligo repigmentation is narrow-band UVB (NBUVB), but how the hair follicle melanocyte precursors are activated by UV light has not been extensively studied. To better understand this process, we developed an application that combined laser capture microdissection and subsequent whole transcriptome RNA sequencing of hair follicle bulge melanocyte precursors and compared their gene signatures to that of regenerated mature epidermal melanocytes from NBUVB-treated vitiligo skin. Using this strategy, we found up-regulation of TNC, GJB6, and THBS1 in the hair follicle bulge melanocytes and of TYR in the epidermal melanocytes of the NBUVB-treated vitiligo skin. We validated these results by quantitative real-time-PCR using NBUVB-treated vitiligo skin and untreated normal skin. We also identified that GLI1, a candidate stem cell-associated gene, is significantly up-regulated in the melanocytes captured from NBUVB-treated vitiligo bulge compared with untreated vitiligo bulge. These signals are potential key players in the activation of bulge melanocyte precursors during vitiligo repigmentation.

Regulation of Head and Neck Squamous Cancer Stem Cells by PI3K and SOX2.

Background: We have an incomplete understanding of the differences between cancer stem cells (CSCs) in human papillomavirus-positive (HPV-positive) and -negative (HPV-negative) head and neck squamous cell cancer (HNSCC). The PI3K pathway has the most frequent activating genetic events in HNSCC (especially HPV-positive driven), but the differential signaling between CSCs and non-CSCs is also unknown. Methods: We addressed these unresolved questions using CSCs identified from 10 HNSCC patient-derived xenografts (PDXs). Sored populations were serially passaged in nude mice to evaluate tumorigenicity and tumor recapitulation. The transcription profile of HNSCC CSCs was characterized by mRNA sequencing, and the susceptibility of CSCs to therapy was investigated using an in vivo model. SOX2 transcriptional activity was used to follow the asymmetric division of PDX-derived CSCs. All statistical tests were two-sided. Results: CSCs were enriched by high aldehyde dehydrogenase (ALDH) activity and CD44 expression and were similar between HPV-positive and HPV-negative cases (percent tumor formation injecting ≤ 1x10(3) cells: ALDH(+)CD44(high) = 65.8%, ALDH(-)CD44(high) = 33.1%, ALDH(+)CD44(high) = 20.0%; and injecting 1x10(5) cells: ALDH(-)CD44(low) = 4.4%). CSCs were resistant to conventional therapy and had PI3K/mTOR pathway overexpression (GSEA pathway enrichment, P < .001), and PI3K inhibition in vivo decreased their tumorigenicity (40.0%-100.0% across cases). PI3K/mTOR directly regulated SOX2 protein levels, and SOX2 in turn activated ALDH1A1 (P < .001 013C and 067C) expression and ALDH activity (ALDH(+) [%] empty-control vs SOX2, 0.4% ± 0.4% vs 14.5% ± 9.8%, P = .03 for 013C and 1.7% ± 1.3% vs 3.6% ± 3.4%, P = .04 for 067C) in 013C and 067 cells. SOX2 enhanced sphere and tumor growth (spheres/well, 013C P < .001 and 067C P = .04) and therapy resistance. SOX2 expression prompted mesenchymal-to-epithelial transition (MET) by inducing CDH1 (013C P = .002, 067C P = .01), followed by asymmetric division and proliferation, which contributed to tumor formation. Conclusions: The molecular link between PI3K activation and CSC properties found in this study provides insights into therapeutic strategies for HNSCC. Constitutive expression of SOX2 in HNSCC cells generates a CSC-like population that enables CSC studies.

Isolating RNA from precursor and mature melanocytes from human vitiligo and normal skin using laser capture microdissection.

To characterize the gene expression profile of regenerated melanocytes in the narrow band UVB (NBUVB)-treated vitiligo epidermis and their precursors in the hair follicle, we present here a strategy of RNA isolation from in situ melanocytes using human frozen skin. We developed a rapid immunostaining protocol using the NKI-beteb antibody, which labels differentiated and precursor melanocytes, followed by fluorescent laser capture microdissection. This technique enabled the direct isolation, from melanocyte and adjacent keratinocyte populations, of satisfactory quality RNA that was successfully amplified and analysed by qRT-PCR. The melanocyte-specific gene transcripts TYR, DCT, TYRP1 and PMEL were significantly upregulated in our NBUVB-treated melanocyte samples as compared with the keratinocyte samples, while keratinocyte-specific genes (KRT5 and KRT14) were expressed significantly higher in the keratinocyte samples as compared with the melanocyte samples. Furthermore, in both NBUVB-treated vitiligo skin and normal skin, when bulge melanocytes were compared with epidermal melanocytes, we found significantly lower expression of melanocyte-specific genes and significantly higher expression of three melanocytic stem cell genes (SOX9, WIF1 and SFRP1), while ALCAM and ALDH1A1 transcripts did not show significant variation. We found significantly higher expression of melanocyte-specific genes in the epidermis of NBUVB-treated vitiligo, as compared to the normal skin. When comparing bulge melanocyte samples from untreated vitiligo, NBUVB-treated vitiligo and normal skin, we did not find significant differences in the expression of melanocyte-specific genes or melanocytic stem cell genes. These techniques offer valuable opportunities to study melanocytes and their precursors in vitiligo and other pigmentation disorders.

Shared gene expression patterns in mesenchymal progenitors derived from lung and epidermis in pulmonary arterial hypertension: identifying key pathways in pulmonary vascular disease.

Rapid access to lung-derived cells from stable subjects is a major challenge in the pulmonary hypertension field, given the relative contraindication of lung biopsy. In these studies, we sought to demonstrate the importance of evaluating a cell type that actively participates in disease processes, as well as the potential to translate these findings to vascular beds in other nonlung tissues, in this instance perivascular skin mesenchymal cells (MCs). We utilized posttransplant or autopsy lung explant-derived cells (ABCG2-expressing mesenchymal progenitor cells [MPCs], fibroblasts) and skin-derived MCs to test the hypothesis that perivascular ABCG2 MPCs derived from pulmonary arterial hypertension (PAH) patient lung and skin would express a gene profile reflective of ongoing vascular dysfunction. By analyzing the genetic signatures and pathways associated with abnormal ABCG2 lung MPC phenotypes during PAH and evaluating them in lung- and skin-derived MCs, we have identified potential predictor genes for detection of PAH as well as a targetable mechanism to restore MPCs and microvascular function. These studies are the first to explore the utility of expanding the study of ABCG2 MPC regulation of the pulmonary microvasculature to the epidermis, in order to identify potential markers for adult lung vascular disease, such as PAH.

A pilot study of cetuximab and the hedgehog inhibitor IPI-926 in recurrent/metastatic head and neck squamous cell carcinoma.

BACKGROUND: This phase 1, dose-finding study determined the safety, maximum tolerated dose (MTD)/recommended phase 2 dose (RP2D), antitumor activity, and molecular correlates of IPI-926, a Hedgehog pathway (HhP) inhibitor, combined with cetuximab in patients with relapsed/metastatic squamous cell carcinoma of the head and neck. PATIENTS AND METHODS: Cetuximab was given with a 400mg/m(2) loading dose followed by 250mg/m(2) weekly. IPI-926 was given daily starting two weeks after cetuximab initiation. A "3+3" study design was used. Prior therapy with cetuximab was allowed. Tumor biopsies occurred prior to cetuximab initiation, prior to IPI-926 initiation, and after treatment with both drugs. RESULTS: Nine patients were enrolled. The RP2D was 160mg, the same as the single-agent IPI-926 MTD. Among 9 treated, 8 evaluable patients, the best responses were 1 partial response (12.5%), 4 stable disease (50%), and 3 disease progressions (37.5%). The median progression free survival was 77days (95% confidence interval 39-156). Decreases in tumor size were seen in both cetuximab-naïve patients (one HPV-positive, one HPV-negative). The most frequent treatment-emergent adverse events were fatigue, muscle cramps, and rash. No DLTs were observed. Tumor shrinkage and progression free survival were associated with intra-tumoral ErbB and HhP gene expression down-regulation during therapy, supporting the preclinical hypothesis. CONCLUSION: Treatment with IPI-926 and cetuximab yielded expected toxicities with signs of anti-tumor activity. Serial tumor biopsies were feasible and revealed proof-of-concept biomarkers.

MicroRNA-203 represses selection and expansion of oncogenic Hras transformed tumor initiating cells.

In many mouse models of skin cancer, only a few tumors typically form even though many cells competent for tumorigenesis receive the same oncogenic stimuli. These observations suggest an active selection process for tumor-initiating cells. Here, we use quantitative mRNA- and miR-Seq to determine the impact of Hras(G12V) on the transcriptome of keratinocytes. We discover that microRNA-203 is downregulated by Hras(G12V). Using a knockout mouse model, we demonstrate that loss of microRNA-203 promotes selection and expansion of tumor-initiating cells. Conversely, restoration of microRNA-203 using an inducible model potently inhibits proliferation of these cells. We comprehensively identify microRNA-203 targets required for Hras-initiated tumorigenesis. These targets include critical regulators of the Ras pathway and essential genes required for cell division. This study establishes a role for the loss of microRNA-203 in promoting selection and expansion of Hras mutated cells and identifies a mechanism through which microRNA-203 antagonizes Hras-mediated tumorigenesis.

Narrow Band Ultraviolet B Treatment for Human Vitiligo Is Associated with Proliferation, Migration, and Differentiation of Melanocyte Precursors.

In vitiligo, the autoimmune destruction of epidermal melanocytes produces white spots that can be repigmented by melanocyte precursors from the hair follicles, following stimulation with UV light. We examined by immunofluorescence the distribution of melanocyte markers (C-KIT, DCT, PAX3, and TYR) coupled with markers of proliferation (KI-67) and migration (MCAM) in precursors and mature melanocytes from the hair follicle and the epidermis of untreated and narrow band UVB (NBUVB)-treated human vitiligo skin. NBUVB was associated with a significant increase in the number of melanocytes in the infundibulum and with restoration of the normal melanocyte population in the epidermis, which was lacking in the untreated vitiligo. We identified several precursor populations (melanocyte stem cells, melanoblasts, and other immature phenotypes), and progressively differentiating melanocytes, some with putative migratory and/or proliferative abilities. The primary melanocyte germ was present in the untreated and treated hair follicle bulge, whereas a possible secondary melanocyte germ composed of C-KIT+ melanocytes was found in the infundibulum and interfollicular epidermis of UV-treated vitiligo. This is an exceptional model for studying the mobilization of melanocyte stem cells in human skin. Improved understanding of this process is essential for designing better treatments for vitiligo, ultimately based on melanocyte stem cell activation and mobilization.

Suppressing AP1 factor signaling in the suprabasal epidermis produces a keratoderma phenotype.

Keratodermas comprise a heterogeneous group of highly debilitating and painful disorders characterized by thickening of the skin with marked hyperkeratosis. Some of these diseases are caused by genetic mutation, whereas other forms are acquired in response to environmental factors. Our understanding of signaling changes that underlie these diseases is limited. In the present study, we describe a keratoderma phenotype in mice in response to suprabasal epidermis-specific inhibition of activator protein 1 transcription factor signaling. These mice develop a severe phenotype characterized by hyperplasia, hyperkeratosis, parakeratosis, and impaired epidermal barrier function. The skin is scaled, constricting bands encircle the tail and digits, the footpads are thickened and scaled, and loricrin staining is markedly reduced in the cornified layers and increased in the nucleus. Features of this phenotype, including nuclear loricrin localization and pseudoainhum (autoamputation), are characteristic of the Vohwinkel syndrome. We confirm that the phenotype develops in a loricrin-null genetic background, indicating that suppressed suprabasal AP1 factor function is sufficient to drive this disease. We also show that the phenotype regresses when suprabasal AP1 factor signaling is restored. Our findings suggest that suppression of AP1 factor signaling in the suprabasal epidermis is a key event in the pathogenesis of keratoderma.

Activation of Nrf2 in keratinocytes causes chloracne (MADISH)-like skin disease in mice.

The transcription factor Nrf2 is a key regulator of the cellular stress response, and pharmacological Nrf2 activation is a promising strategy for skin protection and cancer prevention. We show here that prolonged Nrf2 activation in keratinocytes causes sebaceous gland enlargement and seborrhea in mice due to upregulation of the growth factor epigen, which we identified as a novel Nrf2 target. This was accompanied by thickening and hyperkeratosis of hair follicle infundibula. These abnormalities caused dilatation of infundibula, hair loss, and cyst development upon aging. Upregulation of epigen, secretory leukocyte peptidase inhibitor (Slpi), and small proline-rich protein 2d (Sprr2d) in hair follicles was identified as the likely cause of infundibular acanthosis, hyperkeratosis, and cyst formation. These alterations were highly reminiscent to the phenotype of chloracne/"metabolizing acquired dioxin-induced skin hamartomas" (MADISH) patients. Indeed, SLPI, SPRR2, and epigen were strongly expressed in cysts of MADISH patients and upregulated by dioxin in human keratinocytes in an NRF2-dependent manner. These results identify novel Nrf2 activities in the pilosebaceous unit and point to a role of NRF2 in MADISH pathogenesis.

Isolation and characterization of squamous cell carcinoma-derived stem-like cells: role in tumor formation.

In human epidermis, keratinocyte stem cells (KSC) are characterized by high levels of ß1-integrin, resulting in the rapid adhesion to type IV collagen. Since epithelial tumors originate from KSC, we evaluated the features of rapidly adhering (RAD) keratinocytes derived from primary human squamous cell carcinoma of the skin (cSCC). RAD cells expressed higher levels of survivin, a KSC marker, as compared to non-rapidly adhering (NRAD) cells. Moreover, RAD cells proliferated to a greater extent and were more efficient in forming colonies than NRAD cells. RAD cells also migrated significantly better than NRAD cells. When seeded in a silicone chamber and grafted onto the back skin of NOD SCID mice, RAD cells formed tumors 2-4 fold bigger than those derived from NRAD cells. In tumors derived from RAD cells, the mitotic index was significantly higher than in those derived from NRAD cells, while Ki-67 and survivin expression were more pronounced in RAD tumors. This study suggests that SCC RAD stem cells play a critical role in the formation and development of epithelial tumors.

Epithelial stem cell mutations that promote squamous cell carcinoma metastasis.

Squamous cell carcinomas (SCCs) originate in stratified epithelia, with a small subset becoming metastatic. Epithelial stem cells are targets for driver mutations that give rise to SCCs, but it is unknown whether they contribute to oncogenic multipotency and metastasis. We developed a mouse model of SCC by targeting two frequent genetic mutations in human SCCs, oncogene Kras(G12D) activation and Smad4 deletion, to mouse keratin 15-expressing (K15+) stem cells. We show that transgenic mice developed multilineage tumors, including metastatic SCCs. Among cancer stem cell-enriched (CSC-enriched) populations, those with increased side population (SP) cells correlated with epithelial-mesenchymal transition (EMT) and lung metastasis. We show that microRNA-9 (miR-9) contributed to SP expansion and metastasis, and miR-9 inhibition reduced the number of SP cells and metastasis. Increased miR-9 was detected in metastatic human primary SCCs and SCC metastases, and miR-9-transduced human SCC cells exhibited increased invasion. We identified α-catenin as a predominant miR-9 target. Increased miR-9 in human SCC metastases correlated with α-catenin loss but not E-cadherin loss. Our results demonstrate that stem cells with Kras(G12D) activation and Smad4 depletion can produce tumors that are multipotent and susceptible to EMT and metastasis. Additionally, tumor initiation and metastatic properties of CSCs can be uncoupled, with miR-9 regulating the expansion of metastatic CSCs.

Aurora kinase-A deficiency during skin development impairs cell division and stratification.

Aurora kinase-A (Aurora-A) promotes timely entry into mitosis, centrosome maturation, and formation of bipolar spindles. To address the role of Aurora-A in skin development and homeostasis, we interbred a floxed Aurora-A (Aurora-A(fl)) mouse with the Cre-deleter strain, K14.Cre. Aurora-A(fl/fl);Krt14.Cre (Aurora-A(-/-)) mice died shortly after birth. These mice had translucent skin, and histological evaluation showed that the dorsal skin was very thin and fragile with frank erosions. Although the expression of the basal layer marker keratin 14 and the differentiation marker keratin 1 was evident in Aurora-A(-/-) epidermis, there was a marked reduction in the number of suprabasal layers and basal keratinocytes. Dye exclusion assays also showed defects in barrier function. Unlike wild-type cells, Aurora-A(-/-) basal progenitors were delayed in forming two layers at embryonic day (E)13.5 when embryonic skin begins to stratify. Increased numbers of mitotic cells, apoptotic bodies, and polyploid keratinocytes were evident in Aurora-A(-/-) epidermis, indicating that a deficiency in Aurora-A promotes aberrant mitosis, mitotic slippage, and cell death. Finally, Aurora-A(-/-) keratinocytes displayed centrosomal abnormalities that included centrosomes located at nonapical sites in basal cells. Thus, the deletion of Aurora-A in the developing epidermis alters centrosome function of basal keratinocytes and markedly impairs their ability to divide and stratify.

Mimicking hair disorders by genetic manipulation of organ-cultured human hair follicles.

Human hair follicles can be dissected out of scalp skin and cultured in vitro in defined growth medium. Hair follicle organ cultures have previously been used to investigate the molecular and cellular mechanisms through which various factors regulate the maintenance and cycling of adult hair follicles. In this issue, Samuelov et al. transfected organ-cultured human hair follicles with siRNA nucleotides and suppressed the expression of the endogenous P-cadherin gene in follicular keratinocytes. Knocking down the expression of P-cadherin in hair follicles in vitro recapitulated the hair follicle phenotype observed in patients with hypotrichosis with juvenile macular dystrophy (HJMD) and enabled the authors to establish a cause-effect relationship between loss of P-cadherin and suppression of the canonical Wnt signaling pathway and upregulation of TGFß2 during development of the hair abnormalities observed in HJMD patients.