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Wnt3a modulate self-maintained Muse cell population in human adipose derived stem cells
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Abstracts from the 41st Annual Meeting / Journal of Dermatological Science 86 (2017) e1–e95
by immunostaining and FACS. Various gene expression levels by CD271+ cells were analyzed using real time RT-PCR. We also compared epidermal and dermal CD271+ cell numbers in normal skin and chronic skin ulcer patients. Results and conclusion: Epidermal CD271+ cells were proliferated and migrated from 3 days after wounding. Purified epidermal CD271+ cells expressed higher TGF2 and VEGF␣ transcripts than CD271− cells. Delayed wound healing was observed in the aged mice compared with young mice. During the wound healing process, the peak of dermal CD 271+ cell accumulation was delayed in aged mice compared with young mice. The expression levels of collagen-1, -3, -5, F4-80, EGF, FGF2, TGF1, and IL-1␣ were significantly increased in young mice compared with aged mice. Furthermore, purified dermal CD271+ cells expressed higher FGF2, EGF, PDGFB, and TGF1 gene transcripts than CD271cells. These results suggested that epidermal and dermal CD271+ cells were closely associated with wound healing process by producing various growth factors. Epidermal and dermal CD271+ cells in chronic skin ulcer patients were significantly reduced compared with healthy controls. Thus, both epidermal and dermal stem cells can play an important role in wound healing process. http://dx.doi.org/10.1016/j.jdermsci.2017.02.163 P08-11[C03-6] Wnt3a modulate self-maintained Muse cell population in human adipose derived stem cells Takeshi Yamauchi ∗ , Kenshi Yamasaki, Kenichiro Tsuchiyama, Saaya Koike, Setsuya Aiba Department of Dermatology, Tohoku University Graduate School of Medicine, Miyagi, Japan The canonical Wnt/-catenin pathway governs a myriad of biological processes underlying the development and maintenance of tissue homeostasis, including regulation of stem cell self-renewal, cell proliferation and differentiation. Multilineage-differentiating stress enduring stem (Muse) cells are non-tumorigenic pluripotent stem cells. Muse cells can be isolated as stage specific embryonic antigen 3-positive (SSEA3(+)) cells and exist ca. 3% cell population in mesenchymal tissues. We observed that SSEA3(+) cells could arise from SSEA3-negative non-Muse cells population (SSEA3(−)) in cultured human adipose-derived stem cells (hASC). Because we observed SSEA3(+) cells co-exist with Wnt-positive cells in human skin, we hypothesized that progenitor of Muse cells exist in nonMuse cell population and are promoted to Muse cells by Wnt signals. After 1-week culture of SSEA3(−) cells from hASC, we could isolate 2.57 ± 0.76% (Mean ± SD) of SSEA3(+) cells, whereas addition of Wnt3a increased SSEA3(+) cell population to 4.8-6.6%. In cDNA microarray analysis, pluripotent markers Nanog and BMP4 were increased during the culture of the SSEA3(−) cell population. In contrast, SSEA3(−) cells cultured with Wnt5a for 1 week resulted in decreased SSEA3(+) cells to 0.53 ± 0.12%. These SSEA3(+) cells arisen from SSEA3(−) cell population formed cluster in floating culture and could differentiate into three germ layers, neuron, muscle and hepatocyte. These results suggested that SSEA3(+) Muse cell populations can arise from the non-Muse cells population in hASC and that Wnt pathways, especially Wnt3a canonical signals, augment SSEA3 expression and Muse cells dynamics. The microenvironment of Wnt in adipose tissues may support a niche of Muse cells in skin. http://dx.doi.org/10.1016/j.jdermsci.2017.02.164
P08-12[O2-49] Regulation of scar model cell behaviors: Impacts of sulfated glycosaminoglycans and extracellular epimorphin Tomoatsu Horigome 1,2,∗ , Shinya Takumi 2 , Ayumi Nakashima 2 , Hiroko Yano 2 , Yohei Hirai 1 1 Department of Bioscience, School of Science and Technology, Kwansei Gakuin University, Hyogo, Japan 2 Kobayashi Pharmaceutical Co., Ltd., Japan In skin injuries, dermal fibroblasts produce TGF-beta and grow to form alpha-SMA-positive granulation tissues, incomplete regression of which subsequently causes scar formation in the skin. In this study, we investigated the impacts of glycosaminoglycans (GAGs) and extracellular epimorphin (EPM), both of which have been supposed to be involved in wound healing process, on the behaviors of human scar-derived dermal fibroblasts (HSF). Among GAGs tested, a highly sulfated chondroitin sulfate (CS-E) gave most profound effect on the reduction of TGF-beta-induced alpha-SMA and on the production of active MMPs in HSF. Given that a chondroitin sulfate artificially introduced with more sulfate groups (heparinoid) appeared to elicit similar responses in HSF, we used this heparinoid for further studies as a CS-E substitute. We found that heparinoid leads to cell shape changes in HSF to a spindle-like morphology, in accord with dramatic rearrangement of F-actin. Moreover, down-regulation of alphaSMA and EPM was observed after heparinoid treatment. qRT-PCR and Western-blotting analyses indicated that both on transcription and translation/degradation processes of the alpha-SMA expression were significantly attenuated or promoted by heparinoid or EPM, respectively. On the other hand, we found that EPM’s effects are significantly augmented by heparinoid, implying a complex functional control of EPM by heparinoid. Experiments using the recombinant forms of EPM fragments demonstrated that heparinoid strongly binds to the helix C/SNARE domains, adjacent region to the functional site in EPM. Further analyses to elucidate the functional link between TGF-beta, EPM and GAGs on the regulation of alpha-SMA are now underway.
http://dx.doi.org/10.1016/j.jdermsci.2017.02.165 P08-13[O2-50] Human hair-follicle associated pluripotent (HAP) stem cells differentiate to cardiac muscle cells Netsuko Tohgi 1,∗ , Koya Obama 1 , Yuko Hamada 1 , Nobuko Arakawa 1 , Masateru Yashiro 1 , Sumiyuki Mii 1 , Ryoichi Aki 1 , Robert M. Hoffman 2,3 , Yasuyuki Amoh 1 1
The Department of Dermatology, Kitasato University School of Medicine, Japan 2 AntiCancer, Inc., San Diego, CA, United States 3 Department of Surgery, University of California, CA, United States Hair-follicle-associated pluripotent (HAP) stem cells are nestinpositive and keratin 15-negative and differentiate to neurons, glia, keratinocytes, smooth muscle cells, melanocytes and cardiac muscle cells in vitro. HAP stem cells could effect nerve and spinal cord regeneration in mouse models, differentiating to Schwann cells and neurons. We previously showed that mouse HAP stem cells can differentiate to cardiac muscle cells which aggregate into beating cardiac muscle sheets in DMEM supplemented with iso-
Abstracts from the 41st Annual Meeting / Journal of Dermatological Science 86 (2017) e1–e95
by immunostaining and FACS. Various gene expression levels by CD271+ cells were analyzed using real time RT-PCR. We also compared epidermal and dermal CD271+ cell numbers in normal skin and chronic skin ulcer patients. Results and conclusion: Epidermal CD271+ cells were proliferated and migrated from 3 days after wounding. Purified epidermal CD271+ cells expressed higher TGF2 and VEGF␣ transcripts than CD271− cells. Delayed wound healing was observed in the aged mice compared with young mice. During the wound healing process, the peak of dermal CD 271+ cell accumulation was delayed in aged mice compared with young mice. The expression levels of collagen-1, -3, -5, F4-80, EGF, FGF2, TGF1, and IL-1␣ were significantly increased in young mice compared with aged mice. Furthermore, purified dermal CD271+ cells expressed higher FGF2, EGF, PDGFB, and TGF1 gene transcripts than CD271cells. These results suggested that epidermal and dermal CD271+ cells were closely associated with wound healing process by producing various growth factors. Epidermal and dermal CD271+ cells in chronic skin ulcer patients were significantly reduced compared with healthy controls. Thus, both epidermal and dermal stem cells can play an important role in wound healing process. http://dx.doi.org/10.1016/j.jdermsci.2017.02.163 P08-11[C03-6] Wnt3a modulate self-maintained Muse cell population in human adipose derived stem cells Takeshi Yamauchi ∗ , Kenshi Yamasaki, Kenichiro Tsuchiyama, Saaya Koike, Setsuya Aiba Department of Dermatology, Tohoku University Graduate School of Medicine, Miyagi, Japan The canonical Wnt/-catenin pathway governs a myriad of biological processes underlying the development and maintenance of tissue homeostasis, including regulation of stem cell self-renewal, cell proliferation and differentiation. Multilineage-differentiating stress enduring stem (Muse) cells are non-tumorigenic pluripotent stem cells. Muse cells can be isolated as stage specific embryonic antigen 3-positive (SSEA3(+)) cells and exist ca. 3% cell population in mesenchymal tissues. We observed that SSEA3(+) cells could arise from SSEA3-negative non-Muse cells population (SSEA3(−)) in cultured human adipose-derived stem cells (hASC). Because we observed SSEA3(+) cells co-exist with Wnt-positive cells in human skin, we hypothesized that progenitor of Muse cells exist in nonMuse cell population and are promoted to Muse cells by Wnt signals. After 1-week culture of SSEA3(−) cells from hASC, we could isolate 2.57 ± 0.76% (Mean ± SD) of SSEA3(+) cells, whereas addition of Wnt3a increased SSEA3(+) cell population to 4.8-6.6%. In cDNA microarray analysis, pluripotent markers Nanog and BMP4 were increased during the culture of the SSEA3(−) cell population. In contrast, SSEA3(−) cells cultured with Wnt5a for 1 week resulted in decreased SSEA3(+) cells to 0.53 ± 0.12%. These SSEA3(+) cells arisen from SSEA3(−) cell population formed cluster in floating culture and could differentiate into three germ layers, neuron, muscle and hepatocyte. These results suggested that SSEA3(+) Muse cell populations can arise from the non-Muse cells population in hASC and that Wnt pathways, especially Wnt3a canonical signals, augment SSEA3 expression and Muse cells dynamics. The microenvironment of Wnt in adipose tissues may support a niche of Muse cells in skin. http://dx.doi.org/10.1016/j.jdermsci.2017.02.164
P08-12[O2-49] Regulation of scar model cell behaviors: Impacts of sulfated glycosaminoglycans and extracellular epimorphin Tomoatsu Horigome 1,2,∗ , Shinya Takumi 2 , Ayumi Nakashima 2 , Hiroko Yano 2 , Yohei Hirai 1 1 Department of Bioscience, School of Science and Technology, Kwansei Gakuin University, Hyogo, Japan 2 Kobayashi Pharmaceutical Co., Ltd., Japan In skin injuries, dermal fibroblasts produce TGF-beta and grow to form alpha-SMA-positive granulation tissues, incomplete regression of which subsequently causes scar formation in the skin. In this study, we investigated the impacts of glycosaminoglycans (GAGs) and extracellular epimorphin (EPM), both of which have been supposed to be involved in wound healing process, on the behaviors of human scar-derived dermal fibroblasts (HSF). Among GAGs tested, a highly sulfated chondroitin sulfate (CS-E) gave most profound effect on the reduction of TGF-beta-induced alpha-SMA and on the production of active MMPs in HSF. Given that a chondroitin sulfate artificially introduced with more sulfate groups (heparinoid) appeared to elicit similar responses in HSF, we used this heparinoid for further studies as a CS-E substitute. We found that heparinoid leads to cell shape changes in HSF to a spindle-like morphology, in accord with dramatic rearrangement of F-actin. Moreover, down-regulation of alphaSMA and EPM was observed after heparinoid treatment. qRT-PCR and Western-blotting analyses indicated that both on transcription and translation/degradation processes of the alpha-SMA expression were significantly attenuated or promoted by heparinoid or EPM, respectively. On the other hand, we found that EPM’s effects are significantly augmented by heparinoid, implying a complex functional control of EPM by heparinoid. Experiments using the recombinant forms of EPM fragments demonstrated that heparinoid strongly binds to the helix C/SNARE domains, adjacent region to the functional site in EPM. Further analyses to elucidate the functional link between TGF-beta, EPM and GAGs on the regulation of alpha-SMA are now underway.
http://dx.doi.org/10.1016/j.jdermsci.2017.02.165 P08-13[O2-50] Human hair-follicle associated pluripotent (HAP) stem cells differentiate to cardiac muscle cells Netsuko Tohgi 1,∗ , Koya Obama 1 , Yuko Hamada 1 , Nobuko Arakawa 1 , Masateru Yashiro 1 , Sumiyuki Mii 1 , Ryoichi Aki 1 , Robert M. Hoffman 2,3 , Yasuyuki Amoh 1 1
The Department of Dermatology, Kitasato University School of Medicine, Japan 2 AntiCancer, Inc., San Diego, CA, United States 3 Department of Surgery, University of California, CA, United States Hair-follicle-associated pluripotent (HAP) stem cells are nestinpositive and keratin 15-negative and differentiate to neurons, glia, keratinocytes, smooth muscle cells, melanocytes and cardiac muscle cells in vitro. HAP stem cells could effect nerve and spinal cord regeneration in mouse models, differentiating to Schwann cells and neurons. We previously showed that mouse HAP stem cells can differentiate to cardiac muscle cells which aggregate into beating cardiac muscle sheets in DMEM supplemented with iso-