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088 Understanding the role of ABCA12 in the Pathogenesis of Harlequin Ichthyosis
ABSTRACTS | Epidermal Structure and Function 088
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Understanding the role of ABCA12 in the Pathogenesis of Harlequin Ichthyosis F Enjalbert1, P Dewan1, M Caley1, B Fell1, M Morse3, DP Kelsell1, A Enright2 and EA O’Toole1 1 Cell Biology Cutaneous Research, QMUL, London, United Kingdom, 2 Wellcome Trust Genome Campus, EMBL, Hinxton, Cambridge, United Kingdom and 3 ImmunoInflammation Therapy Area, GSK, Stevenage, United Kingdom ABCA12 is known to be critical for skin barrier integrity. Mutations in this gene cause the most severe form of Autosomal Recessive Congenital Ichthyosis, Harlequin Ichthyosis (HI). HI patients have marked hyperkeratosis at birth with fissuring, leading to life-threatening complications due to increased risk of infection, trans-epidermal water and heat loss. The aim of this study was to identify essential pathways involved in the pathomechanisms of Harlequin Ichthyosis, responsible for aberrant epidermal differentiation. We performed RNA-seq on calcium induced primary keratinocytes with siRNA knockdown of ABCA12 and identified 118 genes significantly down-regulated and 36 genes significantly up-regulated (FDR < 0.05). Functional annotation clustering analysis showed changes in epidermal differentiation, fatty acid metabolism, cytokine and interferon signaling. The suppressor of cytokine signaling 3 (SOCS3), a negative feedback regulator of the JAK-STAT signaling pathway, was 2.5 fold downregulated whereas Interleukin-1 (IL1A and IL1B) were 2 fold increased. To investigate these findings further we engineered an ABCA12 CRISPR-Cas9 knockout keratinocyte cell line and compared this with a HI patient-derived cell line and wild type controls. Alterations in differentiation and lipid profile in the HI OT models were observed, recapitulating the HI epidermis phenotype. We found that phospho-STAT1 (Y701) was strongly upregulated in the HI model compared to control. In HI patient skin, the STAT1 expression pattern was altered compared to control skin. The secretion of IL-1a was increased in the HI model compared to control. Both STAT1 and IL-1 regulate the Nitric Oxide (NO) pathway upregulating transcription of inducible NO synthase (iNOS), which we found to be significantly upregulated in HI skin. These data provide insights into the pathogenesis of HI suggest that the NO signaling pathway may be a possible therapeutic target in this disorder.
MiR-30a is an aged-related microRNA that impairs differentiation and induces apoptosis in human epidermis C Muther1, M Garion1, L Jobeili2, O Damour2, T Nedachi3 and J Lamartine1 1 LBTI UMR5305 CNRS/UCBLI, Lyon, France, 2 Hospices civils de Lyon, Lyon, France and 3 Toyo university, Tokyo, Japan The age-related alterations of epidermis are essentially characterized by a defect in the tissue renewal caused by a reduced keratinocytes proliferation and by a disturbance of their differentiation. However, the mechanisms disturbing epidermal homeostasis during aging remain poorly understood. To go further into this question, we focused on microRNAs (miRNAs), a class of non-coding RNAs known to play a key role in the regulation of epidermal homeostasis. To identify age-related microRNAs, we performed an exhaustive miRNAs microarray expression screen in human primary keratinocytes from young or elderly peoples. This screening revealed many microRNAs modulated by aging including miR-30a, exhibiting both strands overexpressed in aged cells and tissue. We also observed that miR-30a-3p and -5p were significantly induced in a reconstructed skin model mimicking chronological aging. We then constructed a lentiviral vector allowing its inducible and stable overexpression in primary keratinocytes. MiR-30a overexpression lead to a strong impairment of the epidermal differentiation with severe defects of the barrier function in an organotypic culture model. We also observed a significant raise of apoptotic cells in the miR-30a overexpressing epidermis. We then identified several gene targets of miR-30a in keratinocytes, including LOX encoding lysyl oxidase, which plays a role in proliferation/differentiation balance of keratinocytes, IDH1 encoding isocitrate deshydrogenase, a key enzyme of cellular metabolism and AVEN encoding a caspase inhibitor. We confirmed LOX, IDH1 and AVEN direct regulation by miR30a in human keratinocytes and observed their repression in aged skin, allowing a possible link between miR-30a induction and the skin-aging phenotype. Our work revealed a new miRNA actor and deciphered new molecular mechanisms to explain some alterations observed in epidermis during aging, especially those concerning keratinocytes differentiation and apoptotic death.
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The N-terminal domain of Filaggrin 2 co-localizes, binds to and activates the Skin aspartic acid protease in the epidermis of human skin M Donovan1, A Thomas-Collignon2, L Simonetti1, S Desbouis1, C Collin-Djangone´1, C Reverdy3, J Rain3, E Formstecher3 and D Bernard1 1 L’Ore´al R&I, Aulnay sous Bois, France, 2 CNRS, Gif sur Yvette, France and 3 Hybrigenics Services, Paris, France Filaggrin processing is a major biological event in the production of the free amino acid component of Natural Moisturizing Factors (NMF) in skin hydration. Skin aspartic acid protease (SASPase) is one of the key enzymes involved in the early processing of filaggrin during epidermal terminal differentiation. Functional mutations in the SASPase gene have resulted in an impaired stratum corneum barrier function and the onset of dry skin condition (xerosis). In previous studies, yeast two hybrid analyses using SASPase as bait against a human reconstructed skin library identified that the N-terminal domain of filaggrin 2 (Flg2-Nter) strongly bound to the N-terminal fragment of SASPase. This study aimed at deepening our understanding of the regulation of SASPase by Flg2-Nter. Surface Plasmon Resonance analyses confirmed SASPase binding by Flg2-Nter which was unique among related S100 proteins. Confocal immunohistochemical studies in human skin, using specific antibodies to SASPase and Flg2-Nter showed that the two proteins co-localized within the stratum granulosum. Their interaction was confirmed by PLA in cultured cells. Western blots confirmed the presence of Flg2-Nter in all layers of the stratum corneum and epidermis. In vitro enzymatic assays and western blots showed that the Flg2-Nter and a peptide from the EF-Hand of this region enhanced SASPase activity and its auto-activation to its 14 kDa active form. Thus, the data suggests that Flg2-Nter can regulate the auto-activation of SASPase, which may be a key event upstream from filaggrin processing to NMF in the human skin.
5C analysis reveals distinct spatial contact networks between gene-rich and gene-poor TADs at the Epidermal Differentiation Complex in skin epithelial cells K Poterlowicz1, J Yarker1, I Malashchuk1, AN Mardaryev1, MR Gdula2, A sharov3, T KohwiShigematsu4, V Botchkarev1 and MY Fessing1 1 University of Bradford, Bradford, United Kingdom, 2 Biochemistry, University of Oxford, Oxford, United Kingdom, 3 Boston University School of Medicine, Biston, MA and 4 University of California San Francisco, San Francisco, CA Mammalian genomes contain several dozens of large (>0.5 Mbp) gene loci harbouring functionally related genes. Such loci often occupy more than one Topologically Associated Domains (TAD), the principal unit of the spatial genome folding that facilitate chromatin contacts within TADs and limit the contacts between TADs. Epidermal Differentiation Complex (EDC) is a 3.1 Mb locus in the mouse genome harbouring 61 functionally related genes that show lineage-specific activation during epidermal keratinocyte differentiation. To characterize spatial chromatin contact at the EDC containing region in keratinocytes, we used Chromosome Conformation Capture Carbon Copy (5C) technology. 5C data validated by 3DFISH demonstrate the organization of EDC locus into four TADs with distinct spatial interaction patterns based on their gene-rich or gene-poor status. Correlation of the ChIP-seq data for H3K4me1 and H3K27ac with the 5C data revealed that the chromatin interactome of the gene-rich TADs at the EDC locus forms extensive intra- and inter-TAD networks connecting gene promoters and enhancers. The promoter-enhancer interactions are enriched for the binding of epigenetic regulators CTCF, Rad21 and Brg1 at the anchoring regions. In contrast to gene-rich TADs, gene-poor TADs show preferential spatial contacts with each other, do not contain active enhancers and show decreased binding of CTCF, Rad21 and Brg1. Thus, spatial interactions involving gene promoters and enhancers at the multi-TAD EDC locus in skin epithelial cells are not restricted by the TAD boundaries and involve, together with intra-TAD interactions, the extensive contacts between the different gene-rich TADs forming the framework for lineage-specific transcription.
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BNIP3-induced autophagy is required for maintenance of epidermal homeostasis M Moriyama, H Kubo, T Morita, T Hayakawa and H Moriyama Pharmaceutical Research and Technology Institute, Kindai University, Higashi-Osaka, Japan The human skin has an important role in barrier function. Ultraviolet rays (UV) from sunlight exposure can cause cell apoptosis in the skin epidermis, resulting in the disruption of the barrier. Previously, we found that BNIP3 was expressed in the suprabasal layer of the epidermis, where autophagosome formation is normally observed. We also found that BNIP3 was indispensable for autophagy induction and keratinocyte differentiation in human primary epidermal keratinocytes (HPEKs). Intriguingly, addition of an autophagy inhibitor significantly suppressed the BNIP3-stimulated differentiation of keratinocytes, suggesting that BNIP3 plays a crucial role in keratinocyte differentiation by inducing autophagy. In this study, our data suggests that BNIP3 is also important for maintenance of skin epidermis. Intriguingly, although UVB irradiation stimulated BNIP3 expression and cleavage of caspase3, suppression of UVB-induced BNIP3 expression led to further increase in cleaved caspase3 levels. This suggests that BNIP3 has a protective effect against UVB-induced apoptosis in keratinocytes. Furthermore, we found that the accumulation of reactive oxygen species (ROS) by UVB irradiation was sufficient to trigger the activation of JNK and ERK MAPK in HPEKs. In turn, activated MAPK mediated the upregulation of BNIP3 expression. Treatment with an antioxidant reagent or a specific inhibitor of MAPK significantly attenuated the expression of BNIP3 triggered by UVB, followed by the induction of cell death by apoptosis. Furthermore, UVB-induced apoptosis was significantly stimulated by autophagy inhibitor. Moreover, BNIP3 was required for the degradation of dysfunctional mitochondria upon UVB irradiation. These data clearly indicated that BNIP3-induced autophagy, which occurs via UVB-generated ROS-mediated JNK and ERK MAPK activation, has a crucial role in the protection of the skin epidermis against UVB irradiation. Overall, our data provide valuable insights into the role of BNIP3 in the maintenance of epidermal homeostasis.
S208 Journal of Investigative Dermatology (2017), Volume 137
Towards modeling ethnic variations in sebaceous lipid production using human iPSC derived sebocytes A Bobier, B Onteniente and J Maruotti Phenocell, EVRY, France The sebaceous gland is an essential component of the skin: it is composed of sebocytes which secrete sebum out to the skin surface and maintain its normal lubrication. While different models of human sebocytes have been developped over the past decades, they suffer from restrictions, such as limited yield for primary cultures, or abnormal karyotype for immortalized cell lines. On the other hand, human induced pluripotent stem cells (hiPSC) proliferate indefinitely yet retain their ability to differentiate into any cell type. We achieved large scale production of hiPSC derived sebocytes (PCi-SEB) that display many characteristics of native sebocytes including morphology, marker expression and key functions, while maintaining a normal karyotype. Differentiation of PCi-SEB from Caucasian, Asian and African hiPSC lines allowed us to model in vitro differences in lipid production observed between these ethnic groups. PCi-SEB were differentiated from hiPSC cell lines PCi-CAU, PCi-ASI and PCi-AFR following a proprietary protocol. After thawing, PCi-SEB were cultured on fibronectine in sebocyte culture medium (PhenoCULT-SEB). PCi-SEB displayed an epithelial morphology with prominent lipid droplets in the cytoplasm. KRT7 and MUC1 were expressed in 80-90% of the cells, while functional markers such as PPARG, FADS2, SCD and MC5R were also strongly expressed. On the functional levels, PCi-SEB responded by increased lipid production to linoleic acid, arachidonic acid and testosterone stimulation. They had a high d6/d9 ratio and displayed a normal karyotype. Interestingly, PCi-SEB of Caucasian and Asian origin did not significantly differ in lipid production in their basal state, while PCi-SEB of African origin produced 3-4 folds more lipids. Sebocytes differentiated from hiPSC may represent an attractive in vitro tool to model ethnic variations in sebaceous lipid for the development of personalized drugs or skin cares. Further work is required to increase the repertoire of hiPSC lines.
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Understanding the role of ABCA12 in the Pathogenesis of Harlequin Ichthyosis F Enjalbert1, P Dewan1, M Caley1, B Fell1, M Morse3, DP Kelsell1, A Enright2 and EA O’Toole1 1 Cell Biology Cutaneous Research, QMUL, London, United Kingdom, 2 Wellcome Trust Genome Campus, EMBL, Hinxton, Cambridge, United Kingdom and 3 ImmunoInflammation Therapy Area, GSK, Stevenage, United Kingdom ABCA12 is known to be critical for skin barrier integrity. Mutations in this gene cause the most severe form of Autosomal Recessive Congenital Ichthyosis, Harlequin Ichthyosis (HI). HI patients have marked hyperkeratosis at birth with fissuring, leading to life-threatening complications due to increased risk of infection, trans-epidermal water and heat loss. The aim of this study was to identify essential pathways involved in the pathomechanisms of Harlequin Ichthyosis, responsible for aberrant epidermal differentiation. We performed RNA-seq on calcium induced primary keratinocytes with siRNA knockdown of ABCA12 and identified 118 genes significantly down-regulated and 36 genes significantly up-regulated (FDR < 0.05). Functional annotation clustering analysis showed changes in epidermal differentiation, fatty acid metabolism, cytokine and interferon signaling. The suppressor of cytokine signaling 3 (SOCS3), a negative feedback regulator of the JAK-STAT signaling pathway, was 2.5 fold downregulated whereas Interleukin-1 (IL1A and IL1B) were 2 fold increased. To investigate these findings further we engineered an ABCA12 CRISPR-Cas9 knockout keratinocyte cell line and compared this with a HI patient-derived cell line and wild type controls. Alterations in differentiation and lipid profile in the HI OT models were observed, recapitulating the HI epidermis phenotype. We found that phospho-STAT1 (Y701) was strongly upregulated in the HI model compared to control. In HI patient skin, the STAT1 expression pattern was altered compared to control skin. The secretion of IL-1a was increased in the HI model compared to control. Both STAT1 and IL-1 regulate the Nitric Oxide (NO) pathway upregulating transcription of inducible NO synthase (iNOS), which we found to be significantly upregulated in HI skin. These data provide insights into the pathogenesis of HI suggest that the NO signaling pathway may be a possible therapeutic target in this disorder.
MiR-30a is an aged-related microRNA that impairs differentiation and induces apoptosis in human epidermis C Muther1, M Garion1, L Jobeili2, O Damour2, T Nedachi3 and J Lamartine1 1 LBTI UMR5305 CNRS/UCBLI, Lyon, France, 2 Hospices civils de Lyon, Lyon, France and 3 Toyo university, Tokyo, Japan The age-related alterations of epidermis are essentially characterized by a defect in the tissue renewal caused by a reduced keratinocytes proliferation and by a disturbance of their differentiation. However, the mechanisms disturbing epidermal homeostasis during aging remain poorly understood. To go further into this question, we focused on microRNAs (miRNAs), a class of non-coding RNAs known to play a key role in the regulation of epidermal homeostasis. To identify age-related microRNAs, we performed an exhaustive miRNAs microarray expression screen in human primary keratinocytes from young or elderly peoples. This screening revealed many microRNAs modulated by aging including miR-30a, exhibiting both strands overexpressed in aged cells and tissue. We also observed that miR-30a-3p and -5p were significantly induced in a reconstructed skin model mimicking chronological aging. We then constructed a lentiviral vector allowing its inducible and stable overexpression in primary keratinocytes. MiR-30a overexpression lead to a strong impairment of the epidermal differentiation with severe defects of the barrier function in an organotypic culture model. We also observed a significant raise of apoptotic cells in the miR-30a overexpressing epidermis. We then identified several gene targets of miR-30a in keratinocytes, including LOX encoding lysyl oxidase, which plays a role in proliferation/differentiation balance of keratinocytes, IDH1 encoding isocitrate deshydrogenase, a key enzyme of cellular metabolism and AVEN encoding a caspase inhibitor. We confirmed LOX, IDH1 and AVEN direct regulation by miR30a in human keratinocytes and observed their repression in aged skin, allowing a possible link between miR-30a induction and the skin-aging phenotype. Our work revealed a new miRNA actor and deciphered new molecular mechanisms to explain some alterations observed in epidermis during aging, especially those concerning keratinocytes differentiation and apoptotic death.
090
091
The N-terminal domain of Filaggrin 2 co-localizes, binds to and activates the Skin aspartic acid protease in the epidermis of human skin M Donovan1, A Thomas-Collignon2, L Simonetti1, S Desbouis1, C Collin-Djangone´1, C Reverdy3, J Rain3, E Formstecher3 and D Bernard1 1 L’Ore´al R&I, Aulnay sous Bois, France, 2 CNRS, Gif sur Yvette, France and 3 Hybrigenics Services, Paris, France Filaggrin processing is a major biological event in the production of the free amino acid component of Natural Moisturizing Factors (NMF) in skin hydration. Skin aspartic acid protease (SASPase) is one of the key enzymes involved in the early processing of filaggrin during epidermal terminal differentiation. Functional mutations in the SASPase gene have resulted in an impaired stratum corneum barrier function and the onset of dry skin condition (xerosis). In previous studies, yeast two hybrid analyses using SASPase as bait against a human reconstructed skin library identified that the N-terminal domain of filaggrin 2 (Flg2-Nter) strongly bound to the N-terminal fragment of SASPase. This study aimed at deepening our understanding of the regulation of SASPase by Flg2-Nter. Surface Plasmon Resonance analyses confirmed SASPase binding by Flg2-Nter which was unique among related S100 proteins. Confocal immunohistochemical studies in human skin, using specific antibodies to SASPase and Flg2-Nter showed that the two proteins co-localized within the stratum granulosum. Their interaction was confirmed by PLA in cultured cells. Western blots confirmed the presence of Flg2-Nter in all layers of the stratum corneum and epidermis. In vitro enzymatic assays and western blots showed that the Flg2-Nter and a peptide from the EF-Hand of this region enhanced SASPase activity and its auto-activation to its 14 kDa active form. Thus, the data suggests that Flg2-Nter can regulate the auto-activation of SASPase, which may be a key event upstream from filaggrin processing to NMF in the human skin.
5C analysis reveals distinct spatial contact networks between gene-rich and gene-poor TADs at the Epidermal Differentiation Complex in skin epithelial cells K Poterlowicz1, J Yarker1, I Malashchuk1, AN Mardaryev1, MR Gdula2, A sharov3, T KohwiShigematsu4, V Botchkarev1 and MY Fessing1 1 University of Bradford, Bradford, United Kingdom, 2 Biochemistry, University of Oxford, Oxford, United Kingdom, 3 Boston University School of Medicine, Biston, MA and 4 University of California San Francisco, San Francisco, CA Mammalian genomes contain several dozens of large (>0.5 Mbp) gene loci harbouring functionally related genes. Such loci often occupy more than one Topologically Associated Domains (TAD), the principal unit of the spatial genome folding that facilitate chromatin contacts within TADs and limit the contacts between TADs. Epidermal Differentiation Complex (EDC) is a 3.1 Mb locus in the mouse genome harbouring 61 functionally related genes that show lineage-specific activation during epidermal keratinocyte differentiation. To characterize spatial chromatin contact at the EDC containing region in keratinocytes, we used Chromosome Conformation Capture Carbon Copy (5C) technology. 5C data validated by 3DFISH demonstrate the organization of EDC locus into four TADs with distinct spatial interaction patterns based on their gene-rich or gene-poor status. Correlation of the ChIP-seq data for H3K4me1 and H3K27ac with the 5C data revealed that the chromatin interactome of the gene-rich TADs at the EDC locus forms extensive intra- and inter-TAD networks connecting gene promoters and enhancers. The promoter-enhancer interactions are enriched for the binding of epigenetic regulators CTCF, Rad21 and Brg1 at the anchoring regions. In contrast to gene-rich TADs, gene-poor TADs show preferential spatial contacts with each other, do not contain active enhancers and show decreased binding of CTCF, Rad21 and Brg1. Thus, spatial interactions involving gene promoters and enhancers at the multi-TAD EDC locus in skin epithelial cells are not restricted by the TAD boundaries and involve, together with intra-TAD interactions, the extensive contacts between the different gene-rich TADs forming the framework for lineage-specific transcription.
092
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BNIP3-induced autophagy is required for maintenance of epidermal homeostasis M Moriyama, H Kubo, T Morita, T Hayakawa and H Moriyama Pharmaceutical Research and Technology Institute, Kindai University, Higashi-Osaka, Japan The human skin has an important role in barrier function. Ultraviolet rays (UV) from sunlight exposure can cause cell apoptosis in the skin epidermis, resulting in the disruption of the barrier. Previously, we found that BNIP3 was expressed in the suprabasal layer of the epidermis, where autophagosome formation is normally observed. We also found that BNIP3 was indispensable for autophagy induction and keratinocyte differentiation in human primary epidermal keratinocytes (HPEKs). Intriguingly, addition of an autophagy inhibitor significantly suppressed the BNIP3-stimulated differentiation of keratinocytes, suggesting that BNIP3 plays a crucial role in keratinocyte differentiation by inducing autophagy. In this study, our data suggests that BNIP3 is also important for maintenance of skin epidermis. Intriguingly, although UVB irradiation stimulated BNIP3 expression and cleavage of caspase3, suppression of UVB-induced BNIP3 expression led to further increase in cleaved caspase3 levels. This suggests that BNIP3 has a protective effect against UVB-induced apoptosis in keratinocytes. Furthermore, we found that the accumulation of reactive oxygen species (ROS) by UVB irradiation was sufficient to trigger the activation of JNK and ERK MAPK in HPEKs. In turn, activated MAPK mediated the upregulation of BNIP3 expression. Treatment with an antioxidant reagent or a specific inhibitor of MAPK significantly attenuated the expression of BNIP3 triggered by UVB, followed by the induction of cell death by apoptosis. Furthermore, UVB-induced apoptosis was significantly stimulated by autophagy inhibitor. Moreover, BNIP3 was required for the degradation of dysfunctional mitochondria upon UVB irradiation. These data clearly indicated that BNIP3-induced autophagy, which occurs via UVB-generated ROS-mediated JNK and ERK MAPK activation, has a crucial role in the protection of the skin epidermis against UVB irradiation. Overall, our data provide valuable insights into the role of BNIP3 in the maintenance of epidermal homeostasis.
S208 Journal of Investigative Dermatology (2017), Volume 137
Towards modeling ethnic variations in sebaceous lipid production using human iPSC derived sebocytes A Bobier, B Onteniente and J Maruotti Phenocell, EVRY, France The sebaceous gland is an essential component of the skin: it is composed of sebocytes which secrete sebum out to the skin surface and maintain its normal lubrication. While different models of human sebocytes have been developped over the past decades, they suffer from restrictions, such as limited yield for primary cultures, or abnormal karyotype for immortalized cell lines. On the other hand, human induced pluripotent stem cells (hiPSC) proliferate indefinitely yet retain their ability to differentiate into any cell type. We achieved large scale production of hiPSC derived sebocytes (PCi-SEB) that display many characteristics of native sebocytes including morphology, marker expression and key functions, while maintaining a normal karyotype. Differentiation of PCi-SEB from Caucasian, Asian and African hiPSC lines allowed us to model in vitro differences in lipid production observed between these ethnic groups. PCi-SEB were differentiated from hiPSC cell lines PCi-CAU, PCi-ASI and PCi-AFR following a proprietary protocol. After thawing, PCi-SEB were cultured on fibronectine in sebocyte culture medium (PhenoCULT-SEB). PCi-SEB displayed an epithelial morphology with prominent lipid droplets in the cytoplasm. KRT7 and MUC1 were expressed in 80-90% of the cells, while functional markers such as PPARG, FADS2, SCD and MC5R were also strongly expressed. On the functional levels, PCi-SEB responded by increased lipid production to linoleic acid, arachidonic acid and testosterone stimulation. They had a high d6/d9 ratio and displayed a normal karyotype. Interestingly, PCi-SEB of Caucasian and Asian origin did not significantly differ in lipid production in their basal state, while PCi-SEB of African origin produced 3-4 folds more lipids. Sebocytes differentiated from hiPSC may represent an attractive in vitro tool to model ethnic variations in sebaceous lipid for the development of personalized drugs or skin cares. Further work is required to increase the repertoire of hiPSC lines.