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857 Human hair follicle regeneration with trichogenic human dermal papilla precursor cells derived from induced pluripotent stem cells
Skin and Hair Developmental Biology | ABSTRACTS 854
855
CCL5 affects the hair-inductive capacity of three-dimensional (3D)-cultured dermal papilla cells Y Sung, M Kwack and M Jung Kyungpook National University School of Medicine, Daegu, Republic of Korea Alkaline phosphatase (ALP) is a dermal papilla (DP) signature gene and ALP activity is reported to be correlated with the trichogenicity of DP cells. In this study, to elucidate the ALPmediated enhancement of DP cell trichogenicity cells, we analyzed secretory factors that are regulated by ALP expression in human DP spheres, using a cytokine array. We observed that C-C motif ligand 5 (CCL5) was down-regulated by ALP ablation in DP spheres. Since CCL5 was previously reported to be upregulated by OVO homolog-like 1 (OVOL1), which in turn is correlated with the trichogenicity of neonatal dermal cells, we investigated whether CCL5 affects the trichogenicity of human DP spheres. Co-implants of CCL5 small interfering RNA (siRNA)-transfected DP spheres and epidermal cells showed significantly impaired hair follicle induction. Taken together, our data suggest that ALP-regulated expression of CCL5 contributes to the trichogenicity of human DP spheres.
Targeted deletion of crif1 in mouse skin epidermis impairs skin homeostasis J Lee1, J Shin1, D Choi2, K Sohn3, J Kim4, Y Lee1 and C Kim1 1 Chungnam National University, Daejeon, Ch’ungch’ong-namdo, Republic of Korea, 2 Chungnam National University, Daejson, Ch’ungch’ong-namdo, Republic of Korea, 3 Chungnam National University, Daejeon, Ch’ungch’ong-namdo, Republic of Korea and 4 Chungnam National University Hospital, Daejeon, Ch’ungch’ong-namdo, Republic of Korea The skin epidermis, which consists mainly of keratinocytes, acts as a physical barrier to infections by regulating keratinocyte proliferation and differentiation. Hair follicles undergo continuous cycling to produce new one. Therefore, optimum supply of energy from the mitochondria is essential for maintaining skin homeostasis and hair growth. CRIF1is a mitochondrial protein that regulates mitoribosome-mediated synthesis and insertion of mitochondrial oxidative phosphorylation polypeptides into the mitochondrial membrane in mammals. Recent studies reveal that conditional knockout (cKO) of Crif1 in specific tissues of mice induced mitochondrial dysfunction. To determine whether the mitochondrial function of keratinocytes affect skin homeostasis and hair morphogenesis, we generated epidermisspecific Crif1cKO mice. Deletion of Crif1 in epidermis resulted in impaired mitochondrial function and Crif1cKO mice died within a week. Keratinocyte proliferation and differentiation were markedly inhibited in Crif1 cKO mice. Furthermore, hair follicle morphogenesis ofCrif1cKO mice was disrupted by down-regulation of Wnt/b-catenin signaling. These results demonstrate that mitochondrial function in keratinocytes is essential for maintaining epidermal homeostasis and hair follicle morphogenesis.
856
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Topical application of newly developed pseudoceramide improved both skin and hair barrier functions B Park1, D Kim2, K Yoo1, M Kim2, S Kim3, J Lim1, H Shin2, Y Sung4, S Bak4, K Kyong5 and S Jeong6 1 Neopharm Co., Ltd, Daejeon, Taejon-jikhalsi, Republic of Korea, 2 Neopharm Co., Ltd, Daejeon, Republic of Korea, 3 Neopharm, Daejeon, Taejon-jikhalsi, Republic of Korea, 4 Kyungpook National University School of Medicine, Daegu, Republic of Korea, 5 Seowon University, Cheongju, Ch’ungch’ong-bukto, Republic of Korea and 6 Seowon Univ, Daejeon, Taejon-jikhalsi, Republic of Korea Beneficial effects of ceramides or pseudoceramide on skin barrier functions have been repeatedly reported. Most of the studies, however, have focused on the (bio)physical properties of ceramides, such as structural arrangement of stratum corneum (SC) intercellular lipids. Recent studies about the important roles of liposensors in skin homeostasis further implies the possibility of developing a “biological pseudoceramide”. Our previous studies suggested that introduction of unsaturated acyl group can change the inert pseudoceramide into active one, in terms of biological activity. In this study, the biological activities of newly developed pseudoceramide with unsaturated fatty acids, bis-oleamido isopropyl alcohol (BOIP), were investigated. As results, acceleration of epidermal permeability barrier function recovery in acutely disruption model was observed. Increased expression of anti-microbial peptides (AMPs) in cultured human epidermal keratinocytes (NHEKs) was also observed. Interestingly, biomechanical analysis showed that application of Ceramide HS on damaged hair improved the mechanical strength and surface friction. Taken together, these results suggest that the newly developed pseudoceramide, BOIP, have beneficial effects on both skin and hair, which propose a potential use for scalp care products.
Human hair follicle regeneration with trichogenic human dermal papilla precursor cells derived from induced pluripotent stem cells J Kim1, B Kang1, Y Zheng2, S Jo1, K Kim1, G Cotsarelis2 and O Kwon1 1 Department of Dermatology, Seoul National University College of Medicine, Seoul, Seoul-t’ukpyolsi, Republic of Korea and 2 Department of Dermatology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA Dermal papilla precursor cells (DPPCs) in dermal condensates are unipotent cell population which can initiate hair follicle (HF) formation with epidermal placode cells. Intact human dermal papilla cells are known to regenerate de novo HFs with mouse epidermal cells. However, these cells cannot be utilized as human HF regenerative source due to the rapid loss of trichogenic properties upon culture. Here, we differentiated induced pluripotent stem cells (iPSCs) into trichogenic DPPCs via neural crest stem cells (NCSCs). Based on human embryonic HF formation process, we focused to mimic DPPCs at the hair placode stage which is the first recognizable status of HF formation. Interestingly, the syndecan-1 (SDC1) expression, normally expressed in human dermal condensates, was increased, whereas the CD133 expression, normally expressed in iPSCs and NCSCs, was dramatically decreased during the differentiation process. Generated SDC1+CD133- cell population showed higher trichogenic properties and HF regeneration efficiency. Human iPSC-derived DPPCs are capable of generating HF structure when combined with hiPSCs-derived epithelial stem cells in skin reconstitution assays. In conclusion, derivation of DPPCs capable of generating de novo human HF can be a novel treatment possibility for permanent alopecia patients.
858
859
IPSC derived keratinocyte differentiation from reprogrammed blood cells J Jackow, Z Guo, H Wobma, H Abaci, Y Doucet, G Vunjak-Novakovic and AM Christiano Columbia University, New York, NY The ability to create autologous, gene-corrected induced pluripotent stem cell (iPSC)-derived keratinocytes (iPS-KCs) represents a major advance towards treatment of patients with dystrophic epidermolysis bullosa (DEB), a severe blistering disorder of the skin. Several KC differentiation (KC-D) protocols have been reported using fibroblast-derived iPSCs, but variability exists between iPSCs generated from different cells sources and reprogramming methods. We successfully generated iPS-KCs from iPSCs derived from Sendai virus (SeV)infected blood cells using the nonintegrating SeV expressing OCT4, SOX2, cMYC and KLF4. Selected iPSC colonies expressed pluripotency markers, exhibited trilineage differentiation, and maintained a normal karyotype. We then used daily administration of 1 mM RA and 10 ng/ml BMP4 for 6 days for KC-D from iPSCs. In order to determine if the efficiency of KC-D is time-dependent, the iPS-KCs were first kept in culture without passaging for 12, 30 or 60 days, and after 4 passages were subjected to in vitro and ex vivo functional analyses. Only the iPS-KCs that were matured for 60 days (iPS 60-KCs) expressed KRT14 and p63 at levels comparable to those expressed by NHKCs. To test the potential of these cells to differentiate ex vivo, the iPS 60-KCs were used to produce skin equivalents (SEs). The SEs generated from the iPS 60-KCs resembled the architecture of normal skin as demonstrated by expression of KRT10 and loricrin markers for suprabasal and granular layers, respectively. Taken together, these data suggest that iPSCs derived from SeV reprogrammed blood cells can be used to generate KCs that can be differentiated to functional cells after 60 days of maturation in vitro. These findings open new options for autologous cell sourcing in iPSC/CRISPR gene editing approaches aimed at developing innovative stem cell therapies for DEB.
Building a functional niche for hair follicle stem cells T Chen National Institute of Biological Sciences, Beijing, China Long-term adult stem cells sustain tissue regeneration throughout the lifetime of an organism. They were hypothesized to originate from embryonic progenitor cells that acquire long-term self-renewal ability and multipotency at the end of organogenesis. The process through which this is achieved often remains unclear. Here, we discovered that long-term hair follicle stem cells arise from embryonic progenitor cells occupying a niche location that is defined by attenuated Wnt/b-catenin signaling. Hair follicle initiation is marked by placode formation, which depends on the activation of Wnt/b-catenin signaling. Soon afterwards, a region with attenuated Wnt/b-catenin signaling emerges in the upper follicle. Embryonic progenitor cells residing in this region gain expression of adult stem cell markers and become definitive longterm hair follicle stem cells at the end of organogenesis. Attenuation of Wnt/b-catenin signaling is a prerequisite for hair follicle stem cell specification because it suppresses Sox9, which is required for stem cell formation.
www.jidonline.org S147
855
CCL5 affects the hair-inductive capacity of three-dimensional (3D)-cultured dermal papilla cells Y Sung, M Kwack and M Jung Kyungpook National University School of Medicine, Daegu, Republic of Korea Alkaline phosphatase (ALP) is a dermal papilla (DP) signature gene and ALP activity is reported to be correlated with the trichogenicity of DP cells. In this study, to elucidate the ALPmediated enhancement of DP cell trichogenicity cells, we analyzed secretory factors that are regulated by ALP expression in human DP spheres, using a cytokine array. We observed that C-C motif ligand 5 (CCL5) was down-regulated by ALP ablation in DP spheres. Since CCL5 was previously reported to be upregulated by OVO homolog-like 1 (OVOL1), which in turn is correlated with the trichogenicity of neonatal dermal cells, we investigated whether CCL5 affects the trichogenicity of human DP spheres. Co-implants of CCL5 small interfering RNA (siRNA)-transfected DP spheres and epidermal cells showed significantly impaired hair follicle induction. Taken together, our data suggest that ALP-regulated expression of CCL5 contributes to the trichogenicity of human DP spheres.
Targeted deletion of crif1 in mouse skin epidermis impairs skin homeostasis J Lee1, J Shin1, D Choi2, K Sohn3, J Kim4, Y Lee1 and C Kim1 1 Chungnam National University, Daejeon, Ch’ungch’ong-namdo, Republic of Korea, 2 Chungnam National University, Daejson, Ch’ungch’ong-namdo, Republic of Korea, 3 Chungnam National University, Daejeon, Ch’ungch’ong-namdo, Republic of Korea and 4 Chungnam National University Hospital, Daejeon, Ch’ungch’ong-namdo, Republic of Korea The skin epidermis, which consists mainly of keratinocytes, acts as a physical barrier to infections by regulating keratinocyte proliferation and differentiation. Hair follicles undergo continuous cycling to produce new one. Therefore, optimum supply of energy from the mitochondria is essential for maintaining skin homeostasis and hair growth. CRIF1is a mitochondrial protein that regulates mitoribosome-mediated synthesis and insertion of mitochondrial oxidative phosphorylation polypeptides into the mitochondrial membrane in mammals. Recent studies reveal that conditional knockout (cKO) of Crif1 in specific tissues of mice induced mitochondrial dysfunction. To determine whether the mitochondrial function of keratinocytes affect skin homeostasis and hair morphogenesis, we generated epidermisspecific Crif1cKO mice. Deletion of Crif1 in epidermis resulted in impaired mitochondrial function and Crif1cKO mice died within a week. Keratinocyte proliferation and differentiation were markedly inhibited in Crif1 cKO mice. Furthermore, hair follicle morphogenesis ofCrif1cKO mice was disrupted by down-regulation of Wnt/b-catenin signaling. These results demonstrate that mitochondrial function in keratinocytes is essential for maintaining epidermal homeostasis and hair follicle morphogenesis.
856
857
Topical application of newly developed pseudoceramide improved both skin and hair barrier functions B Park1, D Kim2, K Yoo1, M Kim2, S Kim3, J Lim1, H Shin2, Y Sung4, S Bak4, K Kyong5 and S Jeong6 1 Neopharm Co., Ltd, Daejeon, Taejon-jikhalsi, Republic of Korea, 2 Neopharm Co., Ltd, Daejeon, Republic of Korea, 3 Neopharm, Daejeon, Taejon-jikhalsi, Republic of Korea, 4 Kyungpook National University School of Medicine, Daegu, Republic of Korea, 5 Seowon University, Cheongju, Ch’ungch’ong-bukto, Republic of Korea and 6 Seowon Univ, Daejeon, Taejon-jikhalsi, Republic of Korea Beneficial effects of ceramides or pseudoceramide on skin barrier functions have been repeatedly reported. Most of the studies, however, have focused on the (bio)physical properties of ceramides, such as structural arrangement of stratum corneum (SC) intercellular lipids. Recent studies about the important roles of liposensors in skin homeostasis further implies the possibility of developing a “biological pseudoceramide”. Our previous studies suggested that introduction of unsaturated acyl group can change the inert pseudoceramide into active one, in terms of biological activity. In this study, the biological activities of newly developed pseudoceramide with unsaturated fatty acids, bis-oleamido isopropyl alcohol (BOIP), were investigated. As results, acceleration of epidermal permeability barrier function recovery in acutely disruption model was observed. Increased expression of anti-microbial peptides (AMPs) in cultured human epidermal keratinocytes (NHEKs) was also observed. Interestingly, biomechanical analysis showed that application of Ceramide HS on damaged hair improved the mechanical strength and surface friction. Taken together, these results suggest that the newly developed pseudoceramide, BOIP, have beneficial effects on both skin and hair, which propose a potential use for scalp care products.
Human hair follicle regeneration with trichogenic human dermal papilla precursor cells derived from induced pluripotent stem cells J Kim1, B Kang1, Y Zheng2, S Jo1, K Kim1, G Cotsarelis2 and O Kwon1 1 Department of Dermatology, Seoul National University College of Medicine, Seoul, Seoul-t’ukpyolsi, Republic of Korea and 2 Department of Dermatology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA Dermal papilla precursor cells (DPPCs) in dermal condensates are unipotent cell population which can initiate hair follicle (HF) formation with epidermal placode cells. Intact human dermal papilla cells are known to regenerate de novo HFs with mouse epidermal cells. However, these cells cannot be utilized as human HF regenerative source due to the rapid loss of trichogenic properties upon culture. Here, we differentiated induced pluripotent stem cells (iPSCs) into trichogenic DPPCs via neural crest stem cells (NCSCs). Based on human embryonic HF formation process, we focused to mimic DPPCs at the hair placode stage which is the first recognizable status of HF formation. Interestingly, the syndecan-1 (SDC1) expression, normally expressed in human dermal condensates, was increased, whereas the CD133 expression, normally expressed in iPSCs and NCSCs, was dramatically decreased during the differentiation process. Generated SDC1+CD133- cell population showed higher trichogenic properties and HF regeneration efficiency. Human iPSC-derived DPPCs are capable of generating HF structure when combined with hiPSCs-derived epithelial stem cells in skin reconstitution assays. In conclusion, derivation of DPPCs capable of generating de novo human HF can be a novel treatment possibility for permanent alopecia patients.
858
859
IPSC derived keratinocyte differentiation from reprogrammed blood cells J Jackow, Z Guo, H Wobma, H Abaci, Y Doucet, G Vunjak-Novakovic and AM Christiano Columbia University, New York, NY The ability to create autologous, gene-corrected induced pluripotent stem cell (iPSC)-derived keratinocytes (iPS-KCs) represents a major advance towards treatment of patients with dystrophic epidermolysis bullosa (DEB), a severe blistering disorder of the skin. Several KC differentiation (KC-D) protocols have been reported using fibroblast-derived iPSCs, but variability exists between iPSCs generated from different cells sources and reprogramming methods. We successfully generated iPS-KCs from iPSCs derived from Sendai virus (SeV)infected blood cells using the nonintegrating SeV expressing OCT4, SOX2, cMYC and KLF4. Selected iPSC colonies expressed pluripotency markers, exhibited trilineage differentiation, and maintained a normal karyotype. We then used daily administration of 1 mM RA and 10 ng/ml BMP4 for 6 days for KC-D from iPSCs. In order to determine if the efficiency of KC-D is time-dependent, the iPS-KCs were first kept in culture without passaging for 12, 30 or 60 days, and after 4 passages were subjected to in vitro and ex vivo functional analyses. Only the iPS-KCs that were matured for 60 days (iPS 60-KCs) expressed KRT14 and p63 at levels comparable to those expressed by NHKCs. To test the potential of these cells to differentiate ex vivo, the iPS 60-KCs were used to produce skin equivalents (SEs). The SEs generated from the iPS 60-KCs resembled the architecture of normal skin as demonstrated by expression of KRT10 and loricrin markers for suprabasal and granular layers, respectively. Taken together, these data suggest that iPSCs derived from SeV reprogrammed blood cells can be used to generate KCs that can be differentiated to functional cells after 60 days of maturation in vitro. These findings open new options for autologous cell sourcing in iPSC/CRISPR gene editing approaches aimed at developing innovative stem cell therapies for DEB.
Building a functional niche for hair follicle stem cells T Chen National Institute of Biological Sciences, Beijing, China Long-term adult stem cells sustain tissue regeneration throughout the lifetime of an organism. They were hypothesized to originate from embryonic progenitor cells that acquire long-term self-renewal ability and multipotency at the end of organogenesis. The process through which this is achieved often remains unclear. Here, we discovered that long-term hair follicle stem cells arise from embryonic progenitor cells occupying a niche location that is defined by attenuated Wnt/b-catenin signaling. Hair follicle initiation is marked by placode formation, which depends on the activation of Wnt/b-catenin signaling. Soon afterwards, a region with attenuated Wnt/b-catenin signaling emerges in the upper follicle. Embryonic progenitor cells residing in this region gain expression of adult stem cell markers and become definitive longterm hair follicle stem cells at the end of organogenesis. Attenuation of Wnt/b-catenin signaling is a prerequisite for hair follicle stem cell specification because it suppresses Sox9, which is required for stem cell formation.
www.jidonline.org S147