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The relevance of collagen XVII expression in epidermal differentiation and proliferation
Abstracts / Journal of Dermatological Science 84 (2016) e1–e88
P08-07 The relevance of collagen XVII expression in epidermal differentiation and proliferation Mika Watanabe ∗ , Ken Natsuga, Wataru Nishie, Hiroshi Shimizu Department of Dermatology, Hokkaido University Graduate School of Medicine, Sapporo, Japan Collagen XVII (COL17) is a hemidesmosomal protein located at the dermo-epidermal junction. COL17 deficiency causes junctional epidermolysis bullosa, in which hair loss and skin atrophy develop in early adulthood and in which skin fragility is seen. Recently, COL17 has been reported to be critical for the maintenance of hair follicle stem cells (SCs) and melanocyte SCs, although the role of COL17 in keratinocyte differentiation and proliferation has not been fully elucidated. In this study, we aimed to determine how COL17 engages in epidermal differentiation and proliferation. First, cultured normal human epidermal keratinocytes (NHEKs) were stimulated by the EGFR inhibitor AG1478 and bone morphogenetic protein 2/7 (BMP2/7) to induce differentiation. As reported previously, the expression of differentiation markers, including transglutaminase-1, envoplakin and periplakin, was significantly increased, whereas the expression of integrin alpha6 and beta 1 as markers of stemness was reduced in the NHEKs treated with AG1478 and BMP2/7. In line with this, COL17 expression was also diminished in NHEKs treated with AG1478 and BMP2/7, suggesting that keratinocyte differentiation status influences COL17 expression. Next, we performed COL17 knockdown (COL17KD) using siRNA in NHEKs. COL17KD did not alter the expression levels of either stemness or differentiation markers in NHEKs, although the cellular growth rates were decreased in COL17KD NHEKs compared with the mock-transfected cells. Furthermore, COL17-knockout mice showed nail dystrophy before reaching adulthood, which might relate to the low proliferative potential of COL17- deficient keratinocytes. Our data implicate COL17 as playing a pivotal role in the regulation of epidermal keratinocyte differentiation and proliferation. http://dx.doi.org/10.1016/j.jdermsci.2016.08.164 P08-08 Hair-follicle-associated pluripotent (HAP) stem cells Robert M. Hoffman 1,2,∗ , Lingna Li 1 , Sumiyuki Mii 3 , Ryoichi Aki 3 , Jennifer Duong 1 , Aisada Uchugonova 4 , Fang Liu 5 , Wenluo Cao 1,2,5 , Benjamin Tran 1 , Kensei Katsuoka 3 , Yasuyuki Amoh 3 1
AntiCancer Inc., San Diego, CA, United States Department of Surgery, University of California, San Diego, CA, United States 3 Department of Dermatology, Kitasato University School of Medicine, Kanagawa, Japan 4 Department of Biophotonics and Laser Technology, Saarland University, Saarbrueken, Germany 5 Department of Anatomy, Second Military Medical University, Shanghai, China 2
Nestin-expressing cells were originally observed in the upper hair follicle (Proc. Natl. Acad. Sci. USA 100 (2003) 9958–9961). These cells are negative for the keratinocyte marker keratin 15 and can differentiate into neurons, glia, keratinocytes, smooth muscle cells, and melanocytes in vitro (Proc. Natl. Acad. Sci. USA 102 (2005)
e53
5530–5534). We have termed these cells hair-follicle-associatedpluripotent (HAP) stem cells. Mouse and human HAP stem cells can repair the severed sciatic nerve and spinal cord, where they transdifferentiate largely into Schwann cells, and restore nerve and spinal cord function (Proc. Natl. Acad. Sci. USA 102 (2005) 17734–17738; Cell Cycle 7 (2008) 1865–1869). HAP stem cells from both the BA and DP have potential for spinal cord regeneration (Cell Cycle 10 (2011) 830–839). HAP stem cells migrate from the BA to the DP suggesting that the BA may be the source of HAP stem cells of the skin itself (J. Cell. Biochem. 112 (2011) 2046–2050). When mouse whiskers were grown in Gelfoam® histoculture, III tubulin-positive fibers, consisting of HAP stem cells, extended up to 500 mm from the whisker nerve stump. The growing fibers had growth cones on their tips expressing F-actin indicating they were growing axons, suggesting a major function of the HAP stem cells in the hair follicle is for growth of the follicle sensory nerve (J. Cell. Biochem. 114 (2013) 1674–1684). Gelfoam® -cultured HAP stem cells are now being developed for nerve and spinal cord repair. We have now shown that HAP stem cells are readily cyropreserved, indicating that any individual could have a personal bank of cyropreserved HAP stem cells for future needs for nerve or spinal cord repair. Clinical studies are now being planned. http://dx.doi.org/10.1016/j.jdermsci.2016.08.165 P08-09 In vitro mesenchymal–epithelial interaction facilitates de novo hair follicle formation Wei-Hung Wang 1,∗ , Sung-Jan Lin 1,2,3 , Mai-Yi Fan 1 , Chih-Chieh Chan 2 1 Institute of Biomedical Engineering, National Taiwan University, Taipei, Taiwan 2 Department of Dermatology, National Taiwan University Hospital and College of Medicine, Taipei, Taiwan 3 Research Center for Developmental Biology and Regenerative Medicine, National Taiwan University, Taipei, Taiwan
Hair loss is a common clinical condition that usually needs medical treatment or surgical transplant to reduce patient’s psychological stress. However, neither the surgical nor medical treatment can generate new pilosebaceous units to replace the malfunctioned one. In previous works, newborn cells are required to develop de novo hair formation, but the cell source would be a problem in clinical use. So, to establish bioengineering pilosebaceous unit from in vitro expanded cells would be the major task in hair regeneration field, and the most critical problem is the elasticity and availability of adult cells. We hypothesize that the process of forming hair fiber needs the negotiation between keratinocytes and dermal papilla cells. So we use adult dermal papilla cells as an instructive feeder to guide the high-passage adult keratinocytes into differentiation. The educated keratinocytes produce new hair fibers in the subsequent patch assay after co-culture with dermal papilla cells, which implies the instructive cues from mesenchymal–epithelial interaction largely enhance the possibility to promote de novo hair follicle formation. The high-passage keratinocyte, which has been regarded to lose the elasticity, can also be committed to regain their trichogenicity. To analyze the follicular genes expression, such as Krt6 and Krt16, it appears the fate-determination could be decided during co-culture. This finding simply announces the possibility of removing single adult hair, separately expanding dermal and epidermal cells, and then co-culturing these cells before finally mixing them to form new
P08-07 The relevance of collagen XVII expression in epidermal differentiation and proliferation Mika Watanabe ∗ , Ken Natsuga, Wataru Nishie, Hiroshi Shimizu Department of Dermatology, Hokkaido University Graduate School of Medicine, Sapporo, Japan Collagen XVII (COL17) is a hemidesmosomal protein located at the dermo-epidermal junction. COL17 deficiency causes junctional epidermolysis bullosa, in which hair loss and skin atrophy develop in early adulthood and in which skin fragility is seen. Recently, COL17 has been reported to be critical for the maintenance of hair follicle stem cells (SCs) and melanocyte SCs, although the role of COL17 in keratinocyte differentiation and proliferation has not been fully elucidated. In this study, we aimed to determine how COL17 engages in epidermal differentiation and proliferation. First, cultured normal human epidermal keratinocytes (NHEKs) were stimulated by the EGFR inhibitor AG1478 and bone morphogenetic protein 2/7 (BMP2/7) to induce differentiation. As reported previously, the expression of differentiation markers, including transglutaminase-1, envoplakin and periplakin, was significantly increased, whereas the expression of integrin alpha6 and beta 1 as markers of stemness was reduced in the NHEKs treated with AG1478 and BMP2/7. In line with this, COL17 expression was also diminished in NHEKs treated with AG1478 and BMP2/7, suggesting that keratinocyte differentiation status influences COL17 expression. Next, we performed COL17 knockdown (COL17KD) using siRNA in NHEKs. COL17KD did not alter the expression levels of either stemness or differentiation markers in NHEKs, although the cellular growth rates were decreased in COL17KD NHEKs compared with the mock-transfected cells. Furthermore, COL17-knockout mice showed nail dystrophy before reaching adulthood, which might relate to the low proliferative potential of COL17- deficient keratinocytes. Our data implicate COL17 as playing a pivotal role in the regulation of epidermal keratinocyte differentiation and proliferation. http://dx.doi.org/10.1016/j.jdermsci.2016.08.164 P08-08 Hair-follicle-associated pluripotent (HAP) stem cells Robert M. Hoffman 1,2,∗ , Lingna Li 1 , Sumiyuki Mii 3 , Ryoichi Aki 3 , Jennifer Duong 1 , Aisada Uchugonova 4 , Fang Liu 5 , Wenluo Cao 1,2,5 , Benjamin Tran 1 , Kensei Katsuoka 3 , Yasuyuki Amoh 3 1
AntiCancer Inc., San Diego, CA, United States Department of Surgery, University of California, San Diego, CA, United States 3 Department of Dermatology, Kitasato University School of Medicine, Kanagawa, Japan 4 Department of Biophotonics and Laser Technology, Saarland University, Saarbrueken, Germany 5 Department of Anatomy, Second Military Medical University, Shanghai, China 2
Nestin-expressing cells were originally observed in the upper hair follicle (Proc. Natl. Acad. Sci. USA 100 (2003) 9958–9961). These cells are negative for the keratinocyte marker keratin 15 and can differentiate into neurons, glia, keratinocytes, smooth muscle cells, and melanocytes in vitro (Proc. Natl. Acad. Sci. USA 102 (2005)
e53
5530–5534). We have termed these cells hair-follicle-associatedpluripotent (HAP) stem cells. Mouse and human HAP stem cells can repair the severed sciatic nerve and spinal cord, where they transdifferentiate largely into Schwann cells, and restore nerve and spinal cord function (Proc. Natl. Acad. Sci. USA 102 (2005) 17734–17738; Cell Cycle 7 (2008) 1865–1869). HAP stem cells from both the BA and DP have potential for spinal cord regeneration (Cell Cycle 10 (2011) 830–839). HAP stem cells migrate from the BA to the DP suggesting that the BA may be the source of HAP stem cells of the skin itself (J. Cell. Biochem. 112 (2011) 2046–2050). When mouse whiskers were grown in Gelfoam® histoculture, III tubulin-positive fibers, consisting of HAP stem cells, extended up to 500 mm from the whisker nerve stump. The growing fibers had growth cones on their tips expressing F-actin indicating they were growing axons, suggesting a major function of the HAP stem cells in the hair follicle is for growth of the follicle sensory nerve (J. Cell. Biochem. 114 (2013) 1674–1684). Gelfoam® -cultured HAP stem cells are now being developed for nerve and spinal cord repair. We have now shown that HAP stem cells are readily cyropreserved, indicating that any individual could have a personal bank of cyropreserved HAP stem cells for future needs for nerve or spinal cord repair. Clinical studies are now being planned. http://dx.doi.org/10.1016/j.jdermsci.2016.08.165 P08-09 In vitro mesenchymal–epithelial interaction facilitates de novo hair follicle formation Wei-Hung Wang 1,∗ , Sung-Jan Lin 1,2,3 , Mai-Yi Fan 1 , Chih-Chieh Chan 2 1 Institute of Biomedical Engineering, National Taiwan University, Taipei, Taiwan 2 Department of Dermatology, National Taiwan University Hospital and College of Medicine, Taipei, Taiwan 3 Research Center for Developmental Biology and Regenerative Medicine, National Taiwan University, Taipei, Taiwan
Hair loss is a common clinical condition that usually needs medical treatment or surgical transplant to reduce patient’s psychological stress. However, neither the surgical nor medical treatment can generate new pilosebaceous units to replace the malfunctioned one. In previous works, newborn cells are required to develop de novo hair formation, but the cell source would be a problem in clinical use. So, to establish bioengineering pilosebaceous unit from in vitro expanded cells would be the major task in hair regeneration field, and the most critical problem is the elasticity and availability of adult cells. We hypothesize that the process of forming hair fiber needs the negotiation between keratinocytes and dermal papilla cells. So we use adult dermal papilla cells as an instructive feeder to guide the high-passage adult keratinocytes into differentiation. The educated keratinocytes produce new hair fibers in the subsequent patch assay after co-culture with dermal papilla cells, which implies the instructive cues from mesenchymal–epithelial interaction largely enhance the possibility to promote de novo hair follicle formation. The high-passage keratinocyte, which has been regarded to lose the elasticity, can also be committed to regain their trichogenicity. To analyze the follicular genes expression, such as Krt6 and Krt16, it appears the fate-determination could be decided during co-culture. This finding simply announces the possibility of removing single adult hair, separately expanding dermal and epidermal cells, and then co-culturing these cells before finally mixing them to form new