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413 Epidermal dysplasia and hair defects in the disheveled hair and ears (LmnaDhe) mouse
Genetic Disease, Gene Regulation & Gene Therapy | ABSTRACTS 412
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Hairless (Hr) mutations and phenotypes: The new and the old J Cook1, CH Pratt1, D Bikle3, RH Rice2, MV Wiles1 and JP Sundberg1 1 The Jackson Laboratory, Bar Harbor, ME, 2 Environmental Toxicology, University of California, Davis, CA and 3 Dermatology and Medicine, University of California, San Francisco, CA Hairless mice were first described over 150 years ago in the form of the severe rhino mouse mutation (Hrrh) closely followed by a more mild phenotypic allele named hairless (Hrhr). Eventually both these alleles were established in both inbred and outbred mouse colonies. About 30 years ago these mice, primarily the hairless allele as it was called with the milder phenotype, became commonly used for skin cancer research, both UV light and chemical carcinogen induced, as the mice lacked hair as adults and were considered by many to be useful models of normal human skin. Subsequently, it was found that the Hr was a good model of papular atrichia. Today, there is a rich, but possibly confusing, landscape of different mutant Hr alleles present on a multitude of inbred, outbred, congenic, and segregating mouse strains. To confound this problem further there are also a number of mutant Hr phenocopies, i.e. mouse strains with mutations in genes that mimic the hairless phenotype, such as mutant alleles in the Vdr, Odc1, and other genes in the putrescine pathway. In this overview, we will clarify the phenotypes, genotypes, and mouse strain considerations that need to be accounted for when designing a study and interpreting the results of studies using these mice including many new alleles, both spontaneous and genetically engineered.
Epidermal dysplasia and hair defects in the disheveled hair and ears (LmnaDhe) mouse CH Pratt, J Cook and JP Sundberg The Jackson Laboratory, Bar Harbor, ME Lamin A is one component of the nuclear lamina, a chromatin-binding network of type V intermediate filaments, including Lamin C, B1 and B2 which together form a proteinaceous boundary to the nucleus. The Lmna/LMNA gene has over 200 known miss-sense mutations that cause a range of disease phenotypes including progeria, muscular dystrophy, lipodystrophy, and cardiomyopathy. Human patients with mutations in the LMNA gene have a variety of skin abnormalities as does the semi-dominant, spontaneous mutation in the mouse called Disheveled hair and ears (LmnaDhe). Ten day old LmnaDhe/LmnaDhe mice present with a markedly thickened dysplastic epidermis with scattered interfollicular pigmentation, multinucleated kertinocytes, and dystrophic hair follicles when compared to control littermates. The dysplastic epidermis aberrantly expressed mouse specific keratin 6, a nonspecific marker of hyperplasia and neoplasia, associated with downregulation of keratins 1 and 10. The stratum granulosum was thickened with increased expression of filaggrin and loricrin. Expression patterns were normal in wild type and heterozygous mice. Scanning electron microscopy and energy-dispersive X-ray spectroscopy revealed defects in hair shafts associated with low sulfur content of hair in LmnaDhe/LmnaDhe but not in +/+ or +/ LmnaDhe mice. The LmnaDhe mutation in mice causes marked epidermal dysplasia suggesting that mutations in Lmna may play an important role in cutaneous squamous cell carcinoma and possibly other types of skin cancer.
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A new form of ectodermal dysplasia caused by mutations in TSPEAR A Peled1,2, O Sarig1, L Samuelov1,3, M Bertolini4, L Ziv5, D Weissglas-Volkov2, M Eskin-Schwartz1,2, C Adase3, N Malchin1, R Bochner1, G Fainberg1, K Sugawara6, A Baniel1, D Tsuruta6, C Luxemburg2, N Adir7, I Goldberg1, R Gallo3, N Shomron2,8, R Paus4,9 and E Sprecher1,2 1 Tel Aviv Medical Center, Tel Aviv, Israel, 2 Tel-Aviv University, Tel Aviv, Israel, 3 University of California, San Diego, CA, 4 University of Mu¨nster, Mu¨nster, Germany, 5 Sheba Medical Center, Ramat Gan, Israel, 6 Osaka City University, Osaka, Japan, 7 Technion, Haifa, Israel, 8 Variantyx Ltd, Ashland, MA and 9 University of Manchester, Manchester, United Kingdom Despite recent advances in our understanding of the pathogenesis of ectodermal dysplasias (EDs), the molecular basis for many of these disorders remains unknown. Using whole exome sequencing, we identified 2 frameshift and 2 missense mutations in TSPEAR in 3 families affected by a seemingly new form of ED featuring facial dysmorphism, scalp hypotrichosis, body hypertrichosis and hypodontia. Scanning electron microscopy revealed flattened or absent cuticular scales in the patient. Pathway analysis of global gene expression data suggested abnormal NOTCH signaling. Accordingly, TSPEAR down-regulation in primary keratinocytes resulted in decreased NOTCH1 mRNA levels and NOTCH1 protein expression was reduced in patient scalp skin. In addition, TSPEAR knock-down resulted in altered expression of genes known to be regulated by NOTCH and to be involved in hair and tooth development such as TP63 and TGFBR2. TGFBR2 negatively regulates GJA1 while mutations in GJA1 cause the oculo-dento-digital syndrome, which also features prominent hair and dental abnormalities. Interestingly, GJA1 mRNA levels were reduced more than two-fold in TSPEARdeficient keratinocytes. To obtain evidence for an in vivo role of TSPEAR during hair follicle (HF) formation, we used siRNA to knock down the gene in human and K14-H2B GFP mouse HFs. Preliminary data indicated that silencing of TSPEAR inhibits the growth of human HFs and reduces hair bulb diameter, increases HF kinking, and alters the expression of selected hair keratins in mice. In sum, through the study of a rare form of ED, we show that TSPEAR plays a critical role in tooth and hair morphogenesis.
Proteomic analysis of chemokines and cytokines in blister fluids of epidermolysis bullosa patients: Implications for prognosis and therapy V Alexeev1, A Donahue1, J Salas-Alanis2, F Palisson3, J Uitto1 and O Igoucheva1 1 Dermatology and Cutaneous Biology, Thomas Jefferson University, Philadelphia, PA, 2 DebRA Mexico, Guadalupe Nuevo Leo´n, Mexico and 3 DebRa Chile, Avda. Vitacura, Chile The molecular mechanisms that govern directional migration of the stem cells allowing efficient and controlled homing of cells to the skin are poorly understood. The key mechanism that regulates cell recruitment to distal anatomical sites and migration of cells inside affected tissue is chemotaxis, which depends on the signaling molecules termed chemokines. In adult organism, expression of most chemokines is induced in response to physiologic stress or damage. Being secreted, chemokines recruit leukocytes, progenitor stem cells and other cell types to the damaged site as part of host defense and repair mechanism. The striking feature of EB-affected patients is the formation of the intra-epidermal blistering and development of chronic non-healing skin wounds. Analysis of blister fluid can offer the potential to examine local biological mechanisms and define protein signature within lesional skin tissue. Our prior studies demonstrated that adult stem cells express restricted number and relatively low percentage of chemokine receptors, accounting for their limited ability for chemokinedriven migration to the blistering mouse skin. In this study, we conducted comprehensive proteomic screens of chemokines and cytokines in the blister fluids of EB patients to define the inflammatory and immune activities, thus, providing potential markers of disease activity or treatment effect. Also, we investigated the therapeutic relevance of identified chemotactic pathways in vivo providing a basis for future clinical investigations. Collectively, identified molecules provided insight into the mechanisms that govern directional migration and intracutaneous trafficking of systemically infused stem cells, thus, permitting broad and effective application of the therapeutic adult stem cells for EB treatment.
Highly efficient RNA based reprogramming of a small number of human primary fibroblasts G Bilousova1, I Kogut1, SM Ortega1, MV Pavlova1, DP Astling2, K Jones2 and DR Roop1 1 Department of Dermatology, Charles C Gates Center for Regenerative Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO and 2 University of Colorado Anschutz Medical Campus, Aurora, CO Recent advances in reprogramming somatic cells into induced Pluripotent Stem Cells (iPSCs) offer the possibility of developing new stem cell-based approaches for the treatment of inherited skin diseases such as Epidermolysis Bullosa (EB) by coupling homologous recombination with cell reprogramming. Despite the almost limitless therapeutic potential of iPSCs for tissue repair, several obstacles must still be overcome before iPSCs can be applied clinically for the treatment of skin diseases. One of these obstacles is the low efficiency of clinically relevant, integration-free approaches for the reprogramming of patient’s somatic cells into iPSCs. Here, we show that the reprogramming of patient’s fibroblasts into iPSCs can be significantly enhanced via the synergistic activity of reprogramming modified mRNAs and mature miRNA mimics. Our novel, integration- and feeder-free reprogramming approach generates clinically relevant iPSCs from human primary fibroblasts with unprecedented efficiency, reaching 90% on a single cell level. Using this methodology, we have successfully generated iPSCs from fibroblasts isolated from patients with EB simplex and recessive dystrophic EB. All iPSC lines exhibited normal karyotypes and have been successfully maintained for at least 15 passages. The pluripotency of the generated iPSCs was confirmed by gene expression analysis and the differentiation into cell types of all three germ layers both in vitro and in vivo. Thus, our protocol allows for the integration-free reprogramming of somatic human cells with an efficiency and kinetics that surpass all previously published reports. The approach is cost effective, provides an opportunity to shorten the time between the biopsy and the generation of stable high-quality iPSC lines, and allows for the production of iPSCs from individually plated cells in a feeder-free system.
Modulation of microRNAs in skin cell culture models for aging and environmental pollution R Schu¨tz and D Imfeld DSM Nutritional Products Ltd, Kaiseraugst, Switzerland In human skin chronological aging is superimposed by extrinsic aging as a consequence of environmental damage mainly induced by UV light or air pollutants. Epigenetic regulatory mechanisms are key mediators in the aging process that adapt to stressors and age-related changes by imprinting specific epigenetic marks. Age-associated epigenetic changes include alterations in DNA methylation patterns, posttranslational modification of histones, chromatin remodeling, and microRNA expression. MicroRNAs (miRNAs) are short endogenous non-coding RNAs that post-transcriptionally regulate gene expression in many biological processes and can be considered as useful prognostic markers. The development of substances mimicking or modulating miRNA activity would be useful for decreasing undesirable aging effects. The aim of this study was to identify changes in the miRNA expression profiles from skin cell culture models in response to aging or environmental stress as well as upon addition of two potential modulators of miRNA activity. A panel of 105 miRNAs was selected with individually known roles in skin biology and its expression levels were determined by RT-qPCR. The aging model consisted of human epidermal fibroblasts from single adult donors of young, intermediate, and old age. For the pollutant model normal human epidermal keratinocytes were irritated by a single dose of 50 mM benzo(a)pyrene (BaP), a polycyclic aromatic hydrocarbon found in most combustion processes. Two commercial cosmetic bioactives e a polyphenol and a tripeptide - were chosen to analyze the potential of modulating the miRNA expression pattern. For instance miR-150, which is known to modulate keratinocyte differentiation, was significantly down-regulated (7.7 fold change vs vehicle control) in BaP-prestressed cells 24 h after the treatment with polyphenolic test substance. The results showed that miRNA profiles could be linked to age or environmentally stressed changes and miRNA associated functions in skin biology. Furthermore, it offers a new treatment approach for rejuvenating the skin with a variety of bioactives.
www.jidonline.org S73
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Hairless (Hr) mutations and phenotypes: The new and the old J Cook1, CH Pratt1, D Bikle3, RH Rice2, MV Wiles1 and JP Sundberg1 1 The Jackson Laboratory, Bar Harbor, ME, 2 Environmental Toxicology, University of California, Davis, CA and 3 Dermatology and Medicine, University of California, San Francisco, CA Hairless mice were first described over 150 years ago in the form of the severe rhino mouse mutation (Hrrh) closely followed by a more mild phenotypic allele named hairless (Hrhr). Eventually both these alleles were established in both inbred and outbred mouse colonies. About 30 years ago these mice, primarily the hairless allele as it was called with the milder phenotype, became commonly used for skin cancer research, both UV light and chemical carcinogen induced, as the mice lacked hair as adults and were considered by many to be useful models of normal human skin. Subsequently, it was found that the Hr was a good model of papular atrichia. Today, there is a rich, but possibly confusing, landscape of different mutant Hr alleles present on a multitude of inbred, outbred, congenic, and segregating mouse strains. To confound this problem further there are also a number of mutant Hr phenocopies, i.e. mouse strains with mutations in genes that mimic the hairless phenotype, such as mutant alleles in the Vdr, Odc1, and other genes in the putrescine pathway. In this overview, we will clarify the phenotypes, genotypes, and mouse strain considerations that need to be accounted for when designing a study and interpreting the results of studies using these mice including many new alleles, both spontaneous and genetically engineered.
Epidermal dysplasia and hair defects in the disheveled hair and ears (LmnaDhe) mouse CH Pratt, J Cook and JP Sundberg The Jackson Laboratory, Bar Harbor, ME Lamin A is one component of the nuclear lamina, a chromatin-binding network of type V intermediate filaments, including Lamin C, B1 and B2 which together form a proteinaceous boundary to the nucleus. The Lmna/LMNA gene has over 200 known miss-sense mutations that cause a range of disease phenotypes including progeria, muscular dystrophy, lipodystrophy, and cardiomyopathy. Human patients with mutations in the LMNA gene have a variety of skin abnormalities as does the semi-dominant, spontaneous mutation in the mouse called Disheveled hair and ears (LmnaDhe). Ten day old LmnaDhe/LmnaDhe mice present with a markedly thickened dysplastic epidermis with scattered interfollicular pigmentation, multinucleated kertinocytes, and dystrophic hair follicles when compared to control littermates. The dysplastic epidermis aberrantly expressed mouse specific keratin 6, a nonspecific marker of hyperplasia and neoplasia, associated with downregulation of keratins 1 and 10. The stratum granulosum was thickened with increased expression of filaggrin and loricrin. Expression patterns were normal in wild type and heterozygous mice. Scanning electron microscopy and energy-dispersive X-ray spectroscopy revealed defects in hair shafts associated with low sulfur content of hair in LmnaDhe/LmnaDhe but not in +/+ or +/ LmnaDhe mice. The LmnaDhe mutation in mice causes marked epidermal dysplasia suggesting that mutations in Lmna may play an important role in cutaneous squamous cell carcinoma and possibly other types of skin cancer.
414
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417
A new form of ectodermal dysplasia caused by mutations in TSPEAR A Peled1,2, O Sarig1, L Samuelov1,3, M Bertolini4, L Ziv5, D Weissglas-Volkov2, M Eskin-Schwartz1,2, C Adase3, N Malchin1, R Bochner1, G Fainberg1, K Sugawara6, A Baniel1, D Tsuruta6, C Luxemburg2, N Adir7, I Goldberg1, R Gallo3, N Shomron2,8, R Paus4,9 and E Sprecher1,2 1 Tel Aviv Medical Center, Tel Aviv, Israel, 2 Tel-Aviv University, Tel Aviv, Israel, 3 University of California, San Diego, CA, 4 University of Mu¨nster, Mu¨nster, Germany, 5 Sheba Medical Center, Ramat Gan, Israel, 6 Osaka City University, Osaka, Japan, 7 Technion, Haifa, Israel, 8 Variantyx Ltd, Ashland, MA and 9 University of Manchester, Manchester, United Kingdom Despite recent advances in our understanding of the pathogenesis of ectodermal dysplasias (EDs), the molecular basis for many of these disorders remains unknown. Using whole exome sequencing, we identified 2 frameshift and 2 missense mutations in TSPEAR in 3 families affected by a seemingly new form of ED featuring facial dysmorphism, scalp hypotrichosis, body hypertrichosis and hypodontia. Scanning electron microscopy revealed flattened or absent cuticular scales in the patient. Pathway analysis of global gene expression data suggested abnormal NOTCH signaling. Accordingly, TSPEAR down-regulation in primary keratinocytes resulted in decreased NOTCH1 mRNA levels and NOTCH1 protein expression was reduced in patient scalp skin. In addition, TSPEAR knock-down resulted in altered expression of genes known to be regulated by NOTCH and to be involved in hair and tooth development such as TP63 and TGFBR2. TGFBR2 negatively regulates GJA1 while mutations in GJA1 cause the oculo-dento-digital syndrome, which also features prominent hair and dental abnormalities. Interestingly, GJA1 mRNA levels were reduced more than two-fold in TSPEARdeficient keratinocytes. To obtain evidence for an in vivo role of TSPEAR during hair follicle (HF) formation, we used siRNA to knock down the gene in human and K14-H2B GFP mouse HFs. Preliminary data indicated that silencing of TSPEAR inhibits the growth of human HFs and reduces hair bulb diameter, increases HF kinking, and alters the expression of selected hair keratins in mice. In sum, through the study of a rare form of ED, we show that TSPEAR plays a critical role in tooth and hair morphogenesis.
Proteomic analysis of chemokines and cytokines in blister fluids of epidermolysis bullosa patients: Implications for prognosis and therapy V Alexeev1, A Donahue1, J Salas-Alanis2, F Palisson3, J Uitto1 and O Igoucheva1 1 Dermatology and Cutaneous Biology, Thomas Jefferson University, Philadelphia, PA, 2 DebRA Mexico, Guadalupe Nuevo Leo´n, Mexico and 3 DebRa Chile, Avda. Vitacura, Chile The molecular mechanisms that govern directional migration of the stem cells allowing efficient and controlled homing of cells to the skin are poorly understood. The key mechanism that regulates cell recruitment to distal anatomical sites and migration of cells inside affected tissue is chemotaxis, which depends on the signaling molecules termed chemokines. In adult organism, expression of most chemokines is induced in response to physiologic stress or damage. Being secreted, chemokines recruit leukocytes, progenitor stem cells and other cell types to the damaged site as part of host defense and repair mechanism. The striking feature of EB-affected patients is the formation of the intra-epidermal blistering and development of chronic non-healing skin wounds. Analysis of blister fluid can offer the potential to examine local biological mechanisms and define protein signature within lesional skin tissue. Our prior studies demonstrated that adult stem cells express restricted number and relatively low percentage of chemokine receptors, accounting for their limited ability for chemokinedriven migration to the blistering mouse skin. In this study, we conducted comprehensive proteomic screens of chemokines and cytokines in the blister fluids of EB patients to define the inflammatory and immune activities, thus, providing potential markers of disease activity or treatment effect. Also, we investigated the therapeutic relevance of identified chemotactic pathways in vivo providing a basis for future clinical investigations. Collectively, identified molecules provided insight into the mechanisms that govern directional migration and intracutaneous trafficking of systemically infused stem cells, thus, permitting broad and effective application of the therapeutic adult stem cells for EB treatment.
Highly efficient RNA based reprogramming of a small number of human primary fibroblasts G Bilousova1, I Kogut1, SM Ortega1, MV Pavlova1, DP Astling2, K Jones2 and DR Roop1 1 Department of Dermatology, Charles C Gates Center for Regenerative Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO and 2 University of Colorado Anschutz Medical Campus, Aurora, CO Recent advances in reprogramming somatic cells into induced Pluripotent Stem Cells (iPSCs) offer the possibility of developing new stem cell-based approaches for the treatment of inherited skin diseases such as Epidermolysis Bullosa (EB) by coupling homologous recombination with cell reprogramming. Despite the almost limitless therapeutic potential of iPSCs for tissue repair, several obstacles must still be overcome before iPSCs can be applied clinically for the treatment of skin diseases. One of these obstacles is the low efficiency of clinically relevant, integration-free approaches for the reprogramming of patient’s somatic cells into iPSCs. Here, we show that the reprogramming of patient’s fibroblasts into iPSCs can be significantly enhanced via the synergistic activity of reprogramming modified mRNAs and mature miRNA mimics. Our novel, integration- and feeder-free reprogramming approach generates clinically relevant iPSCs from human primary fibroblasts with unprecedented efficiency, reaching 90% on a single cell level. Using this methodology, we have successfully generated iPSCs from fibroblasts isolated from patients with EB simplex and recessive dystrophic EB. All iPSC lines exhibited normal karyotypes and have been successfully maintained for at least 15 passages. The pluripotency of the generated iPSCs was confirmed by gene expression analysis and the differentiation into cell types of all three germ layers both in vitro and in vivo. Thus, our protocol allows for the integration-free reprogramming of somatic human cells with an efficiency and kinetics that surpass all previously published reports. The approach is cost effective, provides an opportunity to shorten the time between the biopsy and the generation of stable high-quality iPSC lines, and allows for the production of iPSCs from individually plated cells in a feeder-free system.
Modulation of microRNAs in skin cell culture models for aging and environmental pollution R Schu¨tz and D Imfeld DSM Nutritional Products Ltd, Kaiseraugst, Switzerland In human skin chronological aging is superimposed by extrinsic aging as a consequence of environmental damage mainly induced by UV light or air pollutants. Epigenetic regulatory mechanisms are key mediators in the aging process that adapt to stressors and age-related changes by imprinting specific epigenetic marks. Age-associated epigenetic changes include alterations in DNA methylation patterns, posttranslational modification of histones, chromatin remodeling, and microRNA expression. MicroRNAs (miRNAs) are short endogenous non-coding RNAs that post-transcriptionally regulate gene expression in many biological processes and can be considered as useful prognostic markers. The development of substances mimicking or modulating miRNA activity would be useful for decreasing undesirable aging effects. The aim of this study was to identify changes in the miRNA expression profiles from skin cell culture models in response to aging or environmental stress as well as upon addition of two potential modulators of miRNA activity. A panel of 105 miRNAs was selected with individually known roles in skin biology and its expression levels were determined by RT-qPCR. The aging model consisted of human epidermal fibroblasts from single adult donors of young, intermediate, and old age. For the pollutant model normal human epidermal keratinocytes were irritated by a single dose of 50 mM benzo(a)pyrene (BaP), a polycyclic aromatic hydrocarbon found in most combustion processes. Two commercial cosmetic bioactives e a polyphenol and a tripeptide - were chosen to analyze the potential of modulating the miRNA expression pattern. For instance miR-150, which is known to modulate keratinocyte differentiation, was significantly down-regulated (7.7 fold change vs vehicle control) in BaP-prestressed cells 24 h after the treatment with polyphenolic test substance. The results showed that miRNA profiles could be linked to age or environmentally stressed changes and miRNA associated functions in skin biology. Furthermore, it offers a new treatment approach for rejuvenating the skin with a variety of bioactives.
www.jidonline.org S73