Salvia officinalis (sage) extract has demonstrated potential as a functional ingredient for skin care application. However, its effect and mechanism in regulating skin pigmentation remain largely unclear. This study investigated the effects of sage ethanol extract (SGE) on melanogenesis and its underlying molecular mechanisms. Treatment with SGE in a human skin equivalent model (3D-skin) suppressed melanin production. To clarify the mechanism of action, the study focused on senescence-associated secretory phenotype (SASP) factors, which are implicated in age-related pigmentation changes. q-PCR and ELISA analyses showed that SGE inhibits melanogenesis by suppressing the expression of granulocyte-macrophage colony-stimulating factor (GM-CSF), a known SASP factor in keratinocytes. Interestingly, a similar effect was observed with L-ascorbic acid 2-glucoside (AG), previously identified as a tyrosinase inhibitor. Importantly, p38 and JNK MAP-kinase were identified as upstream regulators of GM-CSF that are suppressed by SGE. These findings provide new insights into how SGE and AG regulate pigmentation via keratinocyte-derived GM-CSF, highlighting their potential in modulating skin tone and pigmentation through cellular signaling pathways.

The skin plays a protective role against harmful environmental stress such as UV rays. Therefore, the skin is constantly exposed to potential injuries, and wound healing is a vital process for the survival of all higher organisms. Wound healing is dependent on aging and metabolic status at a whole-body level. Because the FOXO family plays a role in aging and metabolism, we investigated the molecular functions of FOXO3A in skin wound healing using FoxO3a-/- mice. We observed that FoxO3a-/- mice showed accelerated skin wound healing. During wound healing, more fibroblasts accumulated at the wound edges and migrated into the wound bed in FoxO3a-/- mice. Moreover, cell migration of dermal fibroblasts isolated from FoxO3a-/- mice was significantly induced. During the in vitro cell migration, we observed accelerated mitochondrial fragmentation and decreased oxygen consumption in the mitochondria of FoxO3a-/- fibroblasts. These changes were caused by the upregulation of mitochondrial Rho GTPase 1, which is an essential mediator of microtubule-based mitochondrial motility. Mitochondrial Rho GTPase 1 inhibition significantly attenuated cell migration, mitochondrial fragmentation, and mitochondrial recruitment to the leading edge of the cells. These data indicate that FOXO3A plays a crucial role in wound healing by regulating mitochondrial dynamics.

Royal jelly (RJ), a natural product secreted by honeybees, is widely used in topical skincare products to help maintain cutaneous homeostasis. Despite its popularity, the mechanism through which RJ exerts its effects on the skin has not been fully elucidated. This study aimed to explore the impact of RJ on the proliferative ability and senescence of human primary epidermal keratinocytes (HPEKs). Our data suggested that epidermal equivalents became thicker with more p63-expressing proliferative cells upon RJ addition to the culture medium. In a two-dimensional culture system, we evaluated the effect of RJ on the proliferation of HPEKs and observed only a slight increase in cell proliferation. This suggests that RJ does not significantly enhance the proliferation of HPEKs in the short term. However, long-term culture experiments demonstrated enhanced population doubling in the RJ-treated group, indicating that RJ inhibits senescence. RJ was found to suppress cellular senescence by modulating the expression levels of ΔNp63, p16, and p21. These results were further supported by the identification of major fatty acids, such as 10-hydroxy-2-decenoic acid, in RJ. Our findings indicate that RJ can maintain epidermal stem cell properties by repressing cellular senescence, providing insights into its mechanism of action in skincare applications.

OBJECTIVE: Human adipose-derived mesenchymal stromal/stem cells (hASC) constitute an attractive source of stem cells for cell-based therapies in regenerative medicine and tissue engineering as they are easy to acquire from lipoaspirate, expansion, and genetic modification ex vivo. The combination of Pdx-1, MafA, and NeuroD1 has been indicated to possess the ability to reprogram various types of cells into insulin-producing cells. The aim of this study is to investigate whether MafA and NeuroD1 would cooperate with Pdx-1 in the differentiation of hASC into insulin-producing cells. MATERIALS AND METHODS: In this experimental study, we generated polycistronic expression vectors expressing Pdx1 and MafA/NeuroD1 with a reporter from a human EF-1α promoter using 2A peptides in a single tet-off lentiviral vector system. Briefly, hASC were transduced with the lentiviral vectors and allowed to differentiate into insulin-producing cells in vitro and in vivo. Thereafter, RNA expression, dithizone staining, and immunofluorescent analysis were conducted. RESULTS: Cleaved transcriptional factors from a single tet-off lentiviral vector were functionally equivalent to their native proteins and strictly regulated by doxycycline (Dox). Insulin gene expression in hASC transduced with Pdx1, Pdx1/ MafA, and Pdx1/NeuroD1 in differentiation medium were successfully increased by 1.89 ± 0.39, 4.81 ± 0.98, 5.51 ± 0.63, respectively, compared to venus-transduced, control hASC. These cells could form dithizone-positive cell clusters in vitro and were found to express insulin in vivo. CONCLUSION: Using our single tet-off lentiviral vector system, Pdx-1 and MafA/NeuroD1 could be simultaneously expressed in the absence of Dox. Further, this system allowed the differentiation of hASC into insulin-producing cells.

Royal jelly (RJ) is secreted by honeybees and has been used as an apitherapy to obtain healthy skin since ancient times. However, the mechanism of the protective effects of RJ against skin aging and skin diseases caused by skin stress and its components have not been clarified. In this study, we attempted to understand the effect of RJ on epidermal function and observed that NAD(P)H quinone dehydrogenase 1 (NQO1) is significantly induced by RJ in keratinocytes. The expression of NQO1 was also increased in the 3D epidermal skin model. NQO1 is involved in antioxidation and detoxification metabolism, and we found that RJ protects against the epidermal stress caused by UVB and menadione through the upregulation of NQO1. We identified 10-hydroxy-2-decenoic acid (10H2DA), a major fatty acid in RJ, as an active compound in this reaction as it induced the expression of NQO1 and protected the skin against oxidative stress. We demonstrated that the protective effect of RJ against epidermal stress is mediated through the upregulation of NQO1 by 10H2DA.

We previously showed that high hydrostatic pressure (HHP) treatment at 200 MPa for 10 min induced complete cell death in skin and skin tumors via necrosis. We used this technique to treat a giant congenital melanocytic nevus and reused the inactivated nevus tissue as a dermis autograft. However, skin inactivated by HHP promoted inflammation in a preclinical study using a porcine model. Therefore, in the present study, we explored the pressurization conditions that induce apoptosis of the skin, as apoptotic cells are not believed to promote inflammation, so the engraftment of inactivated skin should be improved. Using a human dermal fibroblast cell line in suspension culture, we found that HHP at 50 MPa for ≥ 36 h completely induced fibroblast cell death via apoptosis based on the morphological changes in transmission electron microscopy, reactive oxygen species elevation, caspase activation and phosphatidylserine membrane translocation. Furthermore, immunohistochemistry with terminal deoxynucleotidyl transferase dUTP nick-end labeling and cleaved caspase-3 showed most cells in the skin inactivated by pressurization to be apoptotic. Consequently, in vivo grafting of apoptosis-induced inactivated skin resulted in successful engraftment and greater dermal cellular density and macrophage infiltration than our existing method. Our finding supports an alternative approach to hydrostatic pressure application.

High hydrostatic pressure (HHP) is a physical method for inactivating cells or tissues without using chemicals such as detergents. We previously reported that HHP at 200 MPa for 10 min was able to inactivate all cells in skin and giant congenital melanocytic nevus (GCMN) without damaging the extracellular matrix. We also reported that HHP at 150 MPa for 10 min was not sufficient to inactivate them completely, while HHP at 200 MPa for 10 min was able to inactivate them completely. We intend to apply HHP to treat malignant skin tumor as the next step; however, the conditions necessary to kill each kind of cell have not been explored. In this work, we have performed a detailed experimental study on the critical pressure and pressurization time using five kinds of human skin cells and skin tumor cells, including keratinocytes (HEKas), dermal fibroblasts (HDFas), adipose tissue-derived stem cells (ASCs), epidermal melanocytes (HEMa-LPs), and malignant melanoma cells (MMs), using pressures between 150 and 200 MPa. We pressurized cells at 150, 160, 170, 180, or 190 MPa for 1 s, 2 min, and 10 min and evaluated the cellular activity using live/dead staining and proliferation assays. The proliferation assay revealed that HEKas were inactivated at a pressure higher than 150 MPa and a time period longer than 2 min, HDFas and MMs were inactivated at a pressure higher than 160 MPa and for 10 min, and ASCs and HEMa-LPs were inactivated at a pressure higher than 150 MPa and for 10 min. However, some HEMa-LPs were observed alive after HHP at 170 MPa for 10 min, so we concluded that HHP at a pressure higher than 180 MPa for 10 min was able to inactivate five kinds of cells completely.

Wound healing is regulated by complex interactions between the keratinocytes and other cell types including fibroblasts. Recently, adipose-derived mesenchymal stromal/stem cells (ASCs) have been reported to influence wound healing positively via paracrine involvement. However, their roles in keratinocytes are still obscure. Therefore, investigation of the precise effects of ASCs on keratinocytes in an in vitro culture system is required. Our recent data indicate that the epidermal equivalents became thicker on a collagen vitrigel membrane co-cultured with human ASCs (hASCs). Co-culturing the human primary epidermal keratinocytes (HPEK) with hASCs on a collagen vitrigel membrane enhanced their abilities for cell proliferation and adhesion to the membrane but suppressed their differentiation suggesting that hASCs could maintain the undifferentiated status of HPEK. Contrarily, the effects of co-culture using polyethylene terephthalate or polycarbonate membranes for HPEK were completely opposite. These differences may depend on the protein permeability and/or structure of the membrane. Taken together, our data demonstrate that hASCs could be used as a substitute for fibroblasts in skin wound repair, aesthetic medicine, or tissue engineering. It is also important to note that a co-culture system using the collagen vitrigel membrane allows better understanding of the interactions between the keratinocytes and ASCs.

Human adipose-derived mesenchymal stromal cells (hASCs) are attractive for regenerative medicine, but their limited in vitro life span limits their therapeutic applicability. Recent data demonstrate that hypoxia may benefit the ex vivo culture of stem cells. Such cells exhibit a high level of glycolytic metabolism under hypoxic conditions. However, the physiological role of glycolytic activation and its underlying regulatory mechanism are incompletely understood. We have shown that when activated under conditions of 5% O2, Notch signaling dramatically increases the rate of glycolysis, improves proliferation efficiency, prevents senescence, and maintains the multipotency of hASCs. In the present study, we found that activated Notch1 enhanced nuclear p65 levels, resulting in an increase in glucose metabolism through the upregulation of glycolytic factors, including GLUT3. Notch signaling was also involved in glucose metabolism through p53 inactivation. We also found that NF-κB signaling was regulated by p53. These data suggest that Notch-HES1 signaling enhances the glycolytic pathway through p53 and NF-κB. Our data also revealed that activated Notch1 markedly increased the transcriptional activity of hypoxia-inducible factor 1 (HIF-1). Knockdown of HIF-1α significantly attenuated glycolysis induced by activated Notch1, indicating that the glycolysis pathway is regulated by the coordination of Notch signaling and HIF. Overall, our observations provide new regulatory mechanisms for the glycolysis by Notch signaling to maintain the stemness of hASCs.

Here, we carried out a chemical genetic screen using a fission yeast phenotypic assay and showed that ACA-28, a synthetic derivative of 1'-acetoxychavicol acetate (ACA), which is a natural ginger compound, effectively inhibited the growth of melanoma cancer cells wherein ERK MAPK signaling is hyperactivated due to mutations in the upstream activating regulators. ACA-28 more potently inhibited the growth of melanoma cells than did the parental compound ACA. Importantly, the growth of normal human epidermal melanocytes (NHEM) was less affected by ACA-28 at the same 50% inhibitory concentration. In addition, ACA-28 specifically induced apoptosis in NIH/3T3 cells which were oncogenically transformed with human epidermal growth factor receptor-2 (HER2/ErbB2), but not in the parental cells. ACA-28 might serve as a promising seed compound for melanoma treatment.

Genetic modification of human adipose tissue-derived multilineage progenitor cells (hADMPCs) is highly valuable for their exploitation in therapeutic applications. Here, we have developed a novel single tet-off lentiviral vector platform. This vector combines (1) a modified tetracycline (tet)-response element composite promoter, (2) a multi-cistronic strategy to express an improved version of the tet-controlled transactivator and the blasticidin resistance gene under the control of a ubiquitous promoter, and (3) acceptor sites for easy recombination cloning of the gene of interest. In the present study, we used the cytomegalovirus (CMV) or the elongation factor 1 α (EF-1α) promoter as the ubiquitous promoter, and EGFP was introduced as the gene of interest. hADMPCs transduced with a lentiviral vector carrying either the CMV promoter or the EF-1α promoter were effectively selected by blasticidin without affecting their stem cell properties, and EGFP expression was strictly regulated by doxycycline (Dox) treatment in these cells. However, the single tet-off lentiviral vector carrying the EF-1α promoter provided more homogenous expression of EGFP in hADMPCs. Intriguingly, differentiated cells from these Dox-responsive cell lines constitutively expressed EGFP only in the absence of Dox. This single tet-off lentiviral vector thus provides an important tool for applied research on hADMPCs.

Familial hypercholesterolemia (FH) is an autosomal codominant disease characterized by high concentrations of proatherogenic lipoproteins secondary to deficiency in low-density lipoprotein (LDL) receptor. We reported recently the use of in situ stem cell therapy of human adipose tissue-derived multilineage progenitor cells (hADMPCs) in lowering serum total cholesterol in the homozygous Watanabe heritable hyperlipidemic (WHHL) rabbits, an animal model of homozygous FH. Here we demonstrate that pravastatin, an HMG-CoA reductase inhibitor, augmented the cholesterol-lowering effect of transplanted hADMPCs and enhanced LDL clearance in homozygous WHHL rabbit. The results suggest the potential beneficial effects of in situ stem cell therapy in concert with appropriately selected pharmaceutical agents, in regenerative medicine.

To assess the molecular epidemiology of HIV-1 in Republic of Congo (Congo), we investigated 29 HIV-1s obtained from 82 Congolese AIDS and ARC patients in 1996 and 1997. Part of the env region including the V3 loop was phylogenetically analyzed. The genotypes observed were varied: of 29 specimens, 12 (41 %) were subtype A, 1 (3%) was subtype D, 6 (21%) were subtype G, 6 (21%) were subtype H, 2 (7%) were subtype J, and 2 (7%) could not be classified as any known subtypes (U, unclassified). The heterogeneous profile of HIV-1 infection was different from the profiles of neighboring Central African countries. These data show that subtypes G and H as well as subtype A were circulating with high prevalence. The fact that new genetic subtypes (J and U) are circulating indicates a need for a greater surveillance for these subtypes both in Congo as well as in other parts of the world.

In order to assess the incidence of HIV mixed infection as well as to clarify the molecular epidemiology of HIV in central Africa, we investigated 43 HIVs obtained from 211 Cameroonian AC, ARC, and AIDS patients in 1994 and 1995. Part of the pol region and part of the env region were phylogenetically analyzed. The genotypes observed were varied: of 43 specimens, 28 (65%) were subtype A, 1 (2%) was subtype B, 2 (5%) were subtype D, 3 (7%) were subtype F, and 2 (5%) were group O. Of the remaining 7 specimens, 3 were mixed infections with HIV-1 subtypes A and C, HIV-1 subtypes C and F, and HIV-2 subtype A and HIV-1 subtype A; 1 was a mixed infection with HIV-1 subtypes A and D and the highly divergent group O (triple infection); another 3 appeared to consist of mosaic genomes (A/G, A/E, and B/A recombinant). These data show that various types of mixed infection, such as between different subtypes of HIV-1 group M, between HIV-1 and HIV-2, and even between HIV-1 groups O and M, were confirmed at a rather high frequency (approximately 10%). The mixed infection is particularly significant where there is a greater variety of HIV-1 subtypes circulating, since it results in new genetic diversity generated by intersubtype recombination.

Two SHIVs with two or three genes deleted (SHIV-drn and SHIV-dxrn) were constructed. The inoculation of monkeys with SHIV-drn resulted in short-term viremia, but inoculation with SHIV-dxrn did not. At 68 weeks post-inoculation, the monkeys were reinoculated with a 100-fold higher dose of each SHIV, but none showed viremia. Killer cell activities against HIV-1 Env were detected in the SHIV-drn- and SHIV-dxrn-inoculated monkeys. Cross-reactive killer activity against HIV-1 Gag and SIVmac Gag was observed in one monkey. Antibodies were not detected in the SHIV-dxrn-inoculated monkeys, but the SHIV-drn-inoculated monkeys showed an anamnestic antibody reaction. These data indicate that SHIV-drn is infectious to and immuno-inducible in macaques but SHIV-dxrn is not.

本研究は、真の脂肪組織由来間葉系間質幹細胞(hASC)の実像に迫るべく、高解像度のシングルセルレベル解析ならびに性状解析を実施し、未だ未同定のhASCに迫る科学的な成果の探究を目的とする。また、その成果を再生医療に資するhASCの提供（質と量を担保できる橋渡し）としての実用化研究へと連結することも終局目標に据えている。そのため当該年度は、(1) トランスクリプトームレベルでの発現解析と候補遺伝子の峻別を実施し、特徴的な遺伝子発現集団とその遺伝子群のマトランスクリプトームルチオミクス解析から、少なくとも6~7つの亜集団とその遷移または独立関係、ならびに、マーカー候補分子や特異的分化能を有すると思しき遺伝子(群)のヒエラルキーを見出した。また、(2) タンパク質レベルでの候補遺伝子のバリデーション（検証）を行い、(1)で選出した特異的分化能を有すると思しき遺伝子(群)の一部につき、間葉系終末細胞群もしくは外胚葉系終末細胞群への特異的な分化能との関わりが在る事を見出した。さらに、一部の終末細胞については、組織特異的な分化誘導のもと、その機能性についての解析を行い、論文化をすすめた。さらに高度な細胞性状解析を行うため、(3)遺伝子導入系の精査と細胞性状解析のコントロール細胞系列の選定を行い、本解析に至適な、よりhASCの形質維持や分化誘導を制御し易いと思しき遺伝子導入系、ならびに従前に定義されているいわゆるhASを安定した状態で維持できるような培養系の向上、同義に高度な性状解析のためのコントロール細胞(郡)の確立を行った。このよに、総じて本研究は各年度計画を達成するよう、おもだった予備的な結果を紡げていることから、有意義な研究が展開されているものと思われる。

脂肪由来幹細胞の低酸素応答に関わる責任遺伝子群（マスターレギュレータ）を選別し、この特性をもつ細胞亜群が発現する表面マーカーを同定し、間葉系に属する各種の終末細胞への分化特性に傾向があることを見出した。さらに、マスターレギュレータを指標とした脂肪細胞分化に長けたHx-hASCの亜集団の分画は、ある一定の終末細胞に高い分化特性をもつことを見出した。また、人工皮膚3次元モデルの基礎的な検討において、ケラチノサイトなどの上皮構成細胞の維持に対する線維芽細胞との相乗効果、ならびに線維芽細胞とこの脂肪由来間葉系幹細胞亜群との質的量的なバランスが、真皮層のターンオーバーに影響を及ぼすとする知見を得た。

私たちはこれまでに、BNIP3 はオートファジーを介して表皮分化促進効果を持つことを明らかとした。一方、BNIP3 は UVB 照射による アポトーシスから細胞を保護し、皮膚表皮の形態維持に関与していることも明らかとしている。 今回私たちは、UVB照射によるBNIP3の発現は、活性酸素ならびにERK, JNK MAPキナーゼによって制御されていること、BNIP3がオートファジー（マイトファジー）を起こすことで、UVB照射により傷ついたミトコンドリアを分解することでアポトーシスを抑制することを見出した。以上の結果は皮膚炎の治療薬やアンチエイジングに応用出来ることが期待される。

hADSCを、溶存酸素濃度を指標として低酸素培養することにより、新規のhADSCの亜集団を同定した。この時、解糖系が亢されることが認められた。このとき、これらの集団では、低酸素応答因子HIF非依存的なNotchシグナルの活性化が引き起こされていた。Notchシグナルは解糖系亢進に関与するトランスポーター等の発現上昇、解糖系抑制に関与する酵素群の発現抑制に関与することを見出した。また、Notchシグナルはp53シグナル経路の抑制、NF-κBによるGLUT3, TPIの発現上昇に関与していることを明らかにした。同時に、上記の亜集団から、上述の表現型を強く示すさらなる細胞集団の絞り込みに至った。

新規ヒト脂肪組織由来間葉系幹細胞に酸化ストレスを与えると、hADMPCの培養上清がPC12の神経伸張を促すことを見いだしたことから、その機序を調べるために本研究を行った。 まず、hADMPCからグルタチオンを枯渇させると酸化ストレスが生じ、BMP2とFGF2の発現ならびに分泌が上昇することを見いだした。またこの発現・分泌は抗酸化剤であるN-アセチルシステインを添加すると抑制された。さらに、BMP2とFGF2の発現にはp38MAPキナーゼが関与していることが明らかとなった。本研究より、hADMPCを神経変性部位に移植した際、神経伸張を伴う治療効果が期待出来ると考える。