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686 In vitro differentiation of skin-derived precursors into sensory neurons
ABSTRACTS | Wound Healing and Tissue Remodelling 682
683
Development of a 3D living skin equivalent to explore the influence of senescence on the skin ageing phenotype D Milligan1, J Newman1, M Caley1, L Wainwright2, G Jenkins2, M Philpott1 and C Bishop1 1 Cell Biology and Cutaneous Research, The Blizard Institute, London, United Kingdom and 2 Uniliever R&D, Colworth, Bedford, United Kingdom The accumulation of senescent cells with age has been linked to numerous age-associated pathologies, and may be a result of phenotypic changes such the senescence-associated secretory phenotype (SASP), which disrupts tissue homeostasis and can act in a paracrine manner on neighbouring cells. Previously, primary human epithelial cells and fibroblasts were successfully reverted from a deeply senescent (DS) to an early proliferating (EP) phenotype. Comparing EP, DS and senescence reversal cells allowed us to more deeply understand the molecular mechanisms of senescence. To investigate these mechanisms within the context of ageing, a 2D model of senescence has been established by serially passaging human dermal fibroblasts (HDFs) to DS, which display a panel of well-established senescence markers including SA-b-Gal, p16 and p21. HDFs were also induced into premature senescence using UVB and similarly exhibited senescence markers. To examine how senescence contributes to the ageing in a tissue-like environment, a 3D living skin equivalent (LSE) of ageing is in development. EP or mixed EP/DS HDFs are suspended in a dermal matrix while proliferating primary human epidermal keratinocytes are seeded on the surface to create an epidermis. Using this model, we can determine paracrine influences of DS HDFs on the skin phenotype, and, in the future, will investigate if by reversing senescence the skin ageing phenotype can be alleviated. LSEs using EP HDFs show epidermal stratification and spatial expression of proteins corresponding to human skin. High-throughput data extraction platforms have allowed us to quantitatively analyse phenotypic changes between LSEs using immunofluorescence. The latest findings with regards to the contribution of senescent fibroblasts to skin ageing will be presented.
New ex-vivo model containing the 3 layers of the human skin and mimicking fibrosis in adipose tissue R Fitoussi, G Beauchef, P Matarrese and K Vie´ Laboratoires Clarins, Pontoise, France Cellulite appears in hypertrophic fat tissue that is associated with overproduction of extracellular matrix (ECM) proteins. This can induce fibrosis, producing inhomogeneous spatial tension in the tissue. The alteration of the ECM architecture of the adipose tissue can play a fundamental role in the genesis of the fibrotic process. Indeed, this phenomenon is associated with significant changes in the expression of transforming growth factor-b (TGF-b), a profibrosing agent, and metalloproteinases (proteolytic agents degrading the matrix). Moreover, the matrix modification is associated with inflammation and local macrophage infiltration. Finally, local hypoxia during the augmentation in fat mass increases the expression of the hypoxia-inducible factor 1-a (HIF-1a), a factor promoting tissue fibrosis. To study the fibrotic status, we have developed a complete human skin explants model (epidermis / dermis / hypodermis) maintained alive in inflammatory conditions (secretion of medium conditioned by macrophages). To demonstrate the fibrosis level of the adipose tissue, picrosirius red staining was used to detect and estimate collagen deposition in histological sections by microscopy and image analysis. The quantification of collagen deposition was expressed as the ratio of fibrous tissue area stained with picrosirius red/total adipose tissue surface. Under our experimental conditions, we observed the positive impact of the macrophages secretome by a significant picrosirius-red-stained collagen accumulation increase in adipose tissue. Using an original organo-culture model, it was possible to visualize fibrosis on the adipose tissue. Our model can be considered to provide a more realistic picture of what happens in living adipose tissue, in terms of morphology and physiology and a high potential fibrosis model in order to test the efficacy of new active ingredients or creams applied topically.
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Benefits of mineralocorticoid receptor antagonism in cutaneous wound healing V Nguyen1, N Farman1, S Aractingi2 and F Jaisser1 1 Team 1 Mineralocorticoid receptor: pathophysiology and therapeutic innovations, INSERM - U1138 Cordeliers Research Centre, Paris, France and 2 Team: Progenitors and endothelial cells during and after pregnancy, INSERM e UMR S 938, UPMC, Saint Antoine research center, Paris, France Wound repair is a complex biological phenomenon, with interplay between different resident and infiltrating cell types. Impaired wound healing is a major unsolved clinical problem in various situations including patients treated by glucocorticoids and diabetic disease. Efforts towards improving wound healing represent a major challenge, to improve quality of life. In our laboratory, we have questioned the role of mineralocorticoid receptor (MR) in wound healing with proposal that MR blockade could improve this process in pathological situations. Recently, we reported that topical application of MR blockers on the top of the wound can rescue the delayed wound re-epithelialization in glucocorticoid-treated skin and type I diabetic mouse model by restoring the decreased keratinocyte proliferation. In this study, we further investigate the benefit of MR antagonism in a model of type II diabete, the db/db mice. We showed that the db/db mice display a severe delay in wound closure; the mRNA level of MR is higher in wounded skin and the surrounding normal skin of db/db mice than in control mice. Interestingly, topical treatment of MR antagonist Canrenoate could also partly rescue the impaired wound closure in these db/db mice. Altogether, our results demonstrate that MR blockade could rescue at least partly the impairment of wound healing in various pathological situations in mice, including glucocorticoid-treated skin, Type I, and Type II diabetes. Further studies are ongoing to understand the underlying mechanism of the beneficial effect of topical MR antagonism on delayed wound healing.
Locomotive ability of human keratinocyte stem cells is an intrinsic property for stem cell expansion and epidermal reconstruction D Nanba1, F Toki1, H Matsumura1, H Toki2 and EK Nishimura1 1 Department of Stem Cell Biology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan and 2 Research Center for Nuclear Physics, Osaka University, Osaka, Japan Self-organization of tissues from human stem cells in vitro is implicated in regenerative medicine, but the underlying mechanisms are largely unknown. Keratinocyte biology has a great advantage for unveiling tissue reconstruction from stem cells for regenerative medicine because human epidermal keratinocyte stem cells give rise to stratified colonies and generate a stratified squamous epithelium like the epidermis ex vivo. This has achieved the autologous transplantation of reconstructed epidermal sheets onto patients with extensive burns and genetic disorders. Hence, the understanding and regulation of cultured keratinocyte stem cell behavior is essential for successful cell therapy for epidermal defects, which also allows us to better comprehend epidermal homeostasis and regeneration. Here we demonstrate that epidermal reconstruction requires the integration of self-renewing and locomotive ability of human keratinocyte stem cells. Cell kinetic modeling, designed according to the physics of multi-particle systems, predicted that locomotive ability of keratinocyte stem cells is essential for stem cell expansion and the colony growth by reorganizing colony structure. Image analysis revealed that human keratinocytes with higher locomotive ability maintain symmetric cell division and expand the colony. Keratinocytes with limited-growth capacity, however, were less motile and generated the colonies inadequate for epidermal reconstruction. Culture experiments then confirmed that enhancing locomotive ability of keratinocytes results in the sustained colony growth with stem cell expansion. We conclude that locomotive ability of human keratinocyte stem cells is an intrinsic property for stem cell expansion and epidermal reconstruction. This result has important implications in tissue reconstruction and remodeling, and wound healing.
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In vitro differentiation of skin-derived precursors into sensory neurons A Bataille1, R Leschiera1, M Sakka1, E Plee-gautier1, J Carre´1, C Brun2, T Oddos2, L Misery1 and N Lebonvallet1 1 Laboratory Interaction Neuron-Keratinocytes, University of western Brittany, Brest, France and 2 2Johnson & Johnson Sante´ Beaute´ France, Val de Reuil, France Because human sensory neurons (SN), or cell lines, are not available, the culture of SN from animal dorsal root ganglia is commonly used. Studies have highlighted the ability of iPS (induced pluripotent stem cells) or ES (embryonic stem cells) to differentiate into cells with SN characteristics. In most of these studies, cells were differentiated into cells expressing markers of neuronal precursors or cells from the neural crest. In a second step, the induction of Wnt and/or BMP pathways allowed differentiation into SN. Our work aimed to determine if it was possible to directly differentiate human SKP (skin-derived precursors), which are derived from the neural crest and extracted from the skin tissue, into SN. SKPs were extracted by enzymatic and mechanical dissociation of abdominal skin samples. These cells were grown in a DMEM/ F12 medium containing FGF2 and EGF. After few weeks, the cells adhered spontaneously. To induce the Wnt pathway in SKP, we used CHIR99201 that activates the Wnt pathway by inhibiting GS3Kb. For the induction of BMP pathway, we added BMP4. We obtained 1) confirmation by PCR that our SKP cells expressed markers of neural crest and precursors (p75NTR, SOX9, AP2, PAX3) and 2) after differentiation, evidence that they acquired a SN phenotype. Part of the cells also acquired a bipolar neuronal morphology. In qPCR, we observed a 7-fold increase of Brn3a expression (SN marker) after 20 days with CHIR99021 and 8 days of BMP4 compared to undifferentiated cells. In immunochemistry, 100% of cells expressed the neuronal marker neurofilaments and p75NTR, whereas 78 and 75% of cells expressed respectively Brn3a and peripherin (a peripheral neuronal marker). The presence of the TRPV1 channel was also evidenced by immunochemistry and PCR. Altogether, these results demonstrate that SKP cells can be differentiated into cells with a sensory neuron phenotype.
S310 Journal of Investigative Dermatology (2017), Volume 137
Silencing matrix metalloproteinase 1 in epidermal keratinocytes can be beneficial for psoriasis J Mogulevtseva2, A Mezentsev1, S Bruskin1 and ES Piruzian1 1 Functional Genomics, Federal Non-profit Research Institution of Russian Academy of Sciences, NI Vavilov Institute of General Genetics, Moscow, Russian Federation and 2 Agronomy and Biotechnology, KA Timiryazev, Russian Agrarian State University, Moscow, Russian Federation Matrix metalloproteinases are crucial for the maintenance of skin homeostasis, wound healing, and fueling the inflammatory process. In psoriasis, matrix metalloproteinases contribute to epidermal remodeling. They also influence the composition of extracellular matrix and intercellular interactions. Moreover, matrix metalloproteinases are important for vasodilation of dermal microcapillaries. The aim of this paper is to clarify whether knocking matrix metalloproteinase 1 down in epidermal keratinocytes can be beneficial for psoriasis. Lentiviral transduction was used to obtain the cell lines HaCaT-IK and HaCaT-KTR that expressed shRNA specific to matrix metalloproteinase 1 and scrambled shRNA, respectively. Changes in gene expression were analyzed by qPCR. The enzyme activity of matrix metalloproteinase 1 was assessed by zymography. Scratch assay was used to assess migration in transduced cells. Comparative analysis of HaCaT-IK and HaCaT-KTR revealed that MMP1 silencing downregulated matrix metalloproteinase 1 and decreased the enzyme activity of the named enzyme in 7.5 and 4 times, respectively. However, MMP1 silencing changed neither the expression of homologous genes (MMP2, -9 and -12) nor proliferation rate of the transduced cells. Surprisingly, MMP1-silencing changed the expression of several genes that are essential for pathogenesis of psoriasis. Particularly, MMP1-silencing caused downregulation of CCNA2 (0.54 0.14) and KRT17 (0.58 0.18). It also led to induction of LOR (2.08 0.16), FLG (3.29 0.35), KRT1 -5 and -10 (2.06 0.20, 1.50 0.22 and 2.09 0.10, respectively). Finally, MMP1-silencing suppressed cell migration on collagen. In conclusion, MMP1 silencing in human epidermal keratinocytes causes changes in expression of LOR, FLG KRT1, -5, -10 and -17 that can be beneficial for psoriasis.
683
Development of a 3D living skin equivalent to explore the influence of senescence on the skin ageing phenotype D Milligan1, J Newman1, M Caley1, L Wainwright2, G Jenkins2, M Philpott1 and C Bishop1 1 Cell Biology and Cutaneous Research, The Blizard Institute, London, United Kingdom and 2 Uniliever R&D, Colworth, Bedford, United Kingdom The accumulation of senescent cells with age has been linked to numerous age-associated pathologies, and may be a result of phenotypic changes such the senescence-associated secretory phenotype (SASP), which disrupts tissue homeostasis and can act in a paracrine manner on neighbouring cells. Previously, primary human epithelial cells and fibroblasts were successfully reverted from a deeply senescent (DS) to an early proliferating (EP) phenotype. Comparing EP, DS and senescence reversal cells allowed us to more deeply understand the molecular mechanisms of senescence. To investigate these mechanisms within the context of ageing, a 2D model of senescence has been established by serially passaging human dermal fibroblasts (HDFs) to DS, which display a panel of well-established senescence markers including SA-b-Gal, p16 and p21. HDFs were also induced into premature senescence using UVB and similarly exhibited senescence markers. To examine how senescence contributes to the ageing in a tissue-like environment, a 3D living skin equivalent (LSE) of ageing is in development. EP or mixed EP/DS HDFs are suspended in a dermal matrix while proliferating primary human epidermal keratinocytes are seeded on the surface to create an epidermis. Using this model, we can determine paracrine influences of DS HDFs on the skin phenotype, and, in the future, will investigate if by reversing senescence the skin ageing phenotype can be alleviated. LSEs using EP HDFs show epidermal stratification and spatial expression of proteins corresponding to human skin. High-throughput data extraction platforms have allowed us to quantitatively analyse phenotypic changes between LSEs using immunofluorescence. The latest findings with regards to the contribution of senescent fibroblasts to skin ageing will be presented.
New ex-vivo model containing the 3 layers of the human skin and mimicking fibrosis in adipose tissue R Fitoussi, G Beauchef, P Matarrese and K Vie´ Laboratoires Clarins, Pontoise, France Cellulite appears in hypertrophic fat tissue that is associated with overproduction of extracellular matrix (ECM) proteins. This can induce fibrosis, producing inhomogeneous spatial tension in the tissue. The alteration of the ECM architecture of the adipose tissue can play a fundamental role in the genesis of the fibrotic process. Indeed, this phenomenon is associated with significant changes in the expression of transforming growth factor-b (TGF-b), a profibrosing agent, and metalloproteinases (proteolytic agents degrading the matrix). Moreover, the matrix modification is associated with inflammation and local macrophage infiltration. Finally, local hypoxia during the augmentation in fat mass increases the expression of the hypoxia-inducible factor 1-a (HIF-1a), a factor promoting tissue fibrosis. To study the fibrotic status, we have developed a complete human skin explants model (epidermis / dermis / hypodermis) maintained alive in inflammatory conditions (secretion of medium conditioned by macrophages). To demonstrate the fibrosis level of the adipose tissue, picrosirius red staining was used to detect and estimate collagen deposition in histological sections by microscopy and image analysis. The quantification of collagen deposition was expressed as the ratio of fibrous tissue area stained with picrosirius red/total adipose tissue surface. Under our experimental conditions, we observed the positive impact of the macrophages secretome by a significant picrosirius-red-stained collagen accumulation increase in adipose tissue. Using an original organo-culture model, it was possible to visualize fibrosis on the adipose tissue. Our model can be considered to provide a more realistic picture of what happens in living adipose tissue, in terms of morphology and physiology and a high potential fibrosis model in order to test the efficacy of new active ingredients or creams applied topically.
684
685
Benefits of mineralocorticoid receptor antagonism in cutaneous wound healing V Nguyen1, N Farman1, S Aractingi2 and F Jaisser1 1 Team 1 Mineralocorticoid receptor: pathophysiology and therapeutic innovations, INSERM - U1138 Cordeliers Research Centre, Paris, France and 2 Team: Progenitors and endothelial cells during and after pregnancy, INSERM e UMR S 938, UPMC, Saint Antoine research center, Paris, France Wound repair is a complex biological phenomenon, with interplay between different resident and infiltrating cell types. Impaired wound healing is a major unsolved clinical problem in various situations including patients treated by glucocorticoids and diabetic disease. Efforts towards improving wound healing represent a major challenge, to improve quality of life. In our laboratory, we have questioned the role of mineralocorticoid receptor (MR) in wound healing with proposal that MR blockade could improve this process in pathological situations. Recently, we reported that topical application of MR blockers on the top of the wound can rescue the delayed wound re-epithelialization in glucocorticoid-treated skin and type I diabetic mouse model by restoring the decreased keratinocyte proliferation. In this study, we further investigate the benefit of MR antagonism in a model of type II diabete, the db/db mice. We showed that the db/db mice display a severe delay in wound closure; the mRNA level of MR is higher in wounded skin and the surrounding normal skin of db/db mice than in control mice. Interestingly, topical treatment of MR antagonist Canrenoate could also partly rescue the impaired wound closure in these db/db mice. Altogether, our results demonstrate that MR blockade could rescue at least partly the impairment of wound healing in various pathological situations in mice, including glucocorticoid-treated skin, Type I, and Type II diabetes. Further studies are ongoing to understand the underlying mechanism of the beneficial effect of topical MR antagonism on delayed wound healing.
Locomotive ability of human keratinocyte stem cells is an intrinsic property for stem cell expansion and epidermal reconstruction D Nanba1, F Toki1, H Matsumura1, H Toki2 and EK Nishimura1 1 Department of Stem Cell Biology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan and 2 Research Center for Nuclear Physics, Osaka University, Osaka, Japan Self-organization of tissues from human stem cells in vitro is implicated in regenerative medicine, but the underlying mechanisms are largely unknown. Keratinocyte biology has a great advantage for unveiling tissue reconstruction from stem cells for regenerative medicine because human epidermal keratinocyte stem cells give rise to stratified colonies and generate a stratified squamous epithelium like the epidermis ex vivo. This has achieved the autologous transplantation of reconstructed epidermal sheets onto patients with extensive burns and genetic disorders. Hence, the understanding and regulation of cultured keratinocyte stem cell behavior is essential for successful cell therapy for epidermal defects, which also allows us to better comprehend epidermal homeostasis and regeneration. Here we demonstrate that epidermal reconstruction requires the integration of self-renewing and locomotive ability of human keratinocyte stem cells. Cell kinetic modeling, designed according to the physics of multi-particle systems, predicted that locomotive ability of keratinocyte stem cells is essential for stem cell expansion and the colony growth by reorganizing colony structure. Image analysis revealed that human keratinocytes with higher locomotive ability maintain symmetric cell division and expand the colony. Keratinocytes with limited-growth capacity, however, were less motile and generated the colonies inadequate for epidermal reconstruction. Culture experiments then confirmed that enhancing locomotive ability of keratinocytes results in the sustained colony growth with stem cell expansion. We conclude that locomotive ability of human keratinocyte stem cells is an intrinsic property for stem cell expansion and epidermal reconstruction. This result has important implications in tissue reconstruction and remodeling, and wound healing.
686
687
In vitro differentiation of skin-derived precursors into sensory neurons A Bataille1, R Leschiera1, M Sakka1, E Plee-gautier1, J Carre´1, C Brun2, T Oddos2, L Misery1 and N Lebonvallet1 1 Laboratory Interaction Neuron-Keratinocytes, University of western Brittany, Brest, France and 2 2Johnson & Johnson Sante´ Beaute´ France, Val de Reuil, France Because human sensory neurons (SN), or cell lines, are not available, the culture of SN from animal dorsal root ganglia is commonly used. Studies have highlighted the ability of iPS (induced pluripotent stem cells) or ES (embryonic stem cells) to differentiate into cells with SN characteristics. In most of these studies, cells were differentiated into cells expressing markers of neuronal precursors or cells from the neural crest. In a second step, the induction of Wnt and/or BMP pathways allowed differentiation into SN. Our work aimed to determine if it was possible to directly differentiate human SKP (skin-derived precursors), which are derived from the neural crest and extracted from the skin tissue, into SN. SKPs were extracted by enzymatic and mechanical dissociation of abdominal skin samples. These cells were grown in a DMEM/ F12 medium containing FGF2 and EGF. After few weeks, the cells adhered spontaneously. To induce the Wnt pathway in SKP, we used CHIR99201 that activates the Wnt pathway by inhibiting GS3Kb. For the induction of BMP pathway, we added BMP4. We obtained 1) confirmation by PCR that our SKP cells expressed markers of neural crest and precursors (p75NTR, SOX9, AP2, PAX3) and 2) after differentiation, evidence that they acquired a SN phenotype. Part of the cells also acquired a bipolar neuronal morphology. In qPCR, we observed a 7-fold increase of Brn3a expression (SN marker) after 20 days with CHIR99021 and 8 days of BMP4 compared to undifferentiated cells. In immunochemistry, 100% of cells expressed the neuronal marker neurofilaments and p75NTR, whereas 78 and 75% of cells expressed respectively Brn3a and peripherin (a peripheral neuronal marker). The presence of the TRPV1 channel was also evidenced by immunochemistry and PCR. Altogether, these results demonstrate that SKP cells can be differentiated into cells with a sensory neuron phenotype.
S310 Journal of Investigative Dermatology (2017), Volume 137
Silencing matrix metalloproteinase 1 in epidermal keratinocytes can be beneficial for psoriasis J Mogulevtseva2, A Mezentsev1, S Bruskin1 and ES Piruzian1 1 Functional Genomics, Federal Non-profit Research Institution of Russian Academy of Sciences, NI Vavilov Institute of General Genetics, Moscow, Russian Federation and 2 Agronomy and Biotechnology, KA Timiryazev, Russian Agrarian State University, Moscow, Russian Federation Matrix metalloproteinases are crucial for the maintenance of skin homeostasis, wound healing, and fueling the inflammatory process. In psoriasis, matrix metalloproteinases contribute to epidermal remodeling. They also influence the composition of extracellular matrix and intercellular interactions. Moreover, matrix metalloproteinases are important for vasodilation of dermal microcapillaries. The aim of this paper is to clarify whether knocking matrix metalloproteinase 1 down in epidermal keratinocytes can be beneficial for psoriasis. Lentiviral transduction was used to obtain the cell lines HaCaT-IK and HaCaT-KTR that expressed shRNA specific to matrix metalloproteinase 1 and scrambled shRNA, respectively. Changes in gene expression were analyzed by qPCR. The enzyme activity of matrix metalloproteinase 1 was assessed by zymography. Scratch assay was used to assess migration in transduced cells. Comparative analysis of HaCaT-IK and HaCaT-KTR revealed that MMP1 silencing downregulated matrix metalloproteinase 1 and decreased the enzyme activity of the named enzyme in 7.5 and 4 times, respectively. However, MMP1 silencing changed neither the expression of homologous genes (MMP2, -9 and -12) nor proliferation rate of the transduced cells. Surprisingly, MMP1-silencing changed the expression of several genes that are essential for pathogenesis of psoriasis. Particularly, MMP1-silencing caused downregulation of CCNA2 (0.54 0.14) and KRT17 (0.58 0.18). It also led to induction of LOR (2.08 0.16), FLG (3.29 0.35), KRT1 -5 and -10 (2.06 0.20, 1.50 0.22 and 2.09 0.10, respectively). Finally, MMP1-silencing suppressed cell migration on collagen. In conclusion, MMP1 silencing in human epidermal keratinocytes causes changes in expression of LOR, FLG KRT1, -5, -10 and -17 that can be beneficial for psoriasis.