Influence of human induced pluripotent stem cell derived skin organoid-conditioned culture medium on the function of human dermal fibroblasts induced by high glucose
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摘要:
目的 探讨人诱导多能干细胞（iPSC）来源的皮肤类器官条件培养基（SO-CM）对高糖诱导的人真皮成纤维细胞（Fb）功能的影响,为糖尿病创面提供治疗思路。 方法 该研究为实验研究。将人iPSC诱导成皮肤类器官 。 将人iPSC来源的皮肤类器官、人真皮Fb分别接种至皮肤类器官培养基（SOM）培养3 d后,收集细胞培养上清液,分别作为SO-CM和Fb条件培养基（Fb-CM）。采用酶联免疫吸附测定法检测SOM、Fb-CM和SO-CM中肿瘤坏死因子α（TNF-α）、白细胞介素10（IL-10）、IL-18、CC类趋化因子配体2（CCL-2）以及血小板源性生长因子（PDGF）、血管内皮生长因子（VEGF）、转化生长因子β（TGF-β）、表皮生长因子（EGF）、VEGF-β的含量。将高糖诱导的第8、9代人Fb按照随机数字表法分为SOM组、Fb-CM组、SO-CM组,分别使用均含终物质的量浓度35 mmol/L葡萄糖的SOM、Fb-CM、SO-CM培养。培养24 h后,行免疫荧光染色后计算Ki67阳性细胞比,采用细胞计数试剂盒-8检测细胞吸光度值,代表细胞增殖活力；行细胞划痕试验,计算划痕后13 h的细胞迁移率。培养48 h后,采用荧光探针法检测细胞中活性氧表达,采用β-半乳糖苷酶染色试剂盒检测细胞β-半乳糖苷酶阳性染色率（代表细胞衰老情况）。样本数均为3。 结果 3种培养基中TNF-α、IL-18、VEGF-β、PDGF、TGF-β含量比较,差异均无统计学意义（P>0.05）。与SOM相比,Fb-CM和SO-CM中IL-10、EGF含量均明显减少（P<0.05）,Fb-CM中CCL-2含量、SO-CM中VEGF含量均明显增加（P<0.05）。培养24 h后,Fb-CM组和SO-CM组的Ki67阳性细胞比[（45.2±6.0）%、（57.4±4.0）%]和吸光度值（124±5、158±12）均明显高于SOM组[（29.6±2.1）%、100±6,P<0.05],SO-CM组的Ki67阳性细胞比和细胞吸光度值均明显高于Fb-CM组（P<0.05）。划痕后13 h,Fb-CM组、SO-CM组细胞迁移率均明显高于SOM组（P<0.05）。培养48 h后,SO-CM组细胞活性氧水平明显高于SOM组、Fb-CM组（P值均<0.05）。培养48 h后,3组细胞β-半乳糖苷酶阳性染色率组间总体比较,差异无统计学意义（P>0.05）。 结论 SO-CM中VEGF含量高,并能明显促进高糖诱导的人真皮Fb的增殖、迁移和活性氧表达,但对细胞衰老无明显影响。 Abstract:Objective To explore the influence of human induced pluripotent stem cell (iPSC) derived skin organoid-conditioned culture medium (SO-CM) on the function of human dermal fibroblasts (Fbs) induced by high glucose, with the aim of providing treatment ideas for diabetic wounds. Methods This study was an experimental research. Human iPSCs were induced into skin organoids. Human iPSC-derived skin organoids and human dermal Fbs were seeded into skin organoid culture medium (SOM) and cultured for three days, respectively. Then the cell culture supernatants were collected as SO-CM and Fb-conditioned culture medium (Fb-CM), respectively. The content of tumor necrosis factor-α (TNF-α), interleukin-10 (IL-10), IL-18, C-C motif chemokine ligand 2 (CCL-2), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), transforming growth factor-β (TGF-β), epidermal growth factor (EGF), and VEGF-β in SOM, Fb-CM, and SO-CM was detected by enzyme-linked immunosorbent assay. Human Fbs of passage 8 and 9 induced by high glucose were divided into SOM group, Fb-CM group, and SO-CM group according to the random number table method, and were cultured with SOM, Fb-CM, and SO-CM all containing glucose at final molarity of 35 mmol/L, respectively. After 24 hours of culture, the Ki67 positive ratio of cells was calculated after immunofluorescence staining, and the cell absorbance value was detected by using cell counting kit-8, representing cell proliferation activity. The cell scratch test was performed to calculate the cell migration rate at 13 hours after scratching. After 48 hours of culture, the expression of reactive oxygen species in cells was detected by fluorescent probe method, and the rate of β-galactosidase-positive staining of cells was detected by β-galactosidase staining kit, representing cellular senescence. The sample size was three. Results There was no statistically significant difference in the content of TNF-α, PDGF, and TGF-β among the three culture media (P>0.05). Compared with that in SOM, the content of IL-10 and EGF in Fb-CM and SO-CM was significantly decreased (P<0.05), while the content of CCL-2 in FB-CM and VEGF in SO-CM was significantly increased (P<0.05). After 24 hours of culture, the Ki67 positive ratio ((45.2±6.0)% and (57.4±4.0)%) and absorbance values (124±5 and 158±12) of cells in the Fb-CM group and the SO-CM group were significantly higher than those in the SOM group ((29.6±2.1)% and 100±6, P<0.05), and the Ki67 positive ratio and absorbance value of cells in the SO-CM group were significantly higher than those in the Fb-CM group (P<0.05). At 13 hours after scratching, the cell migration rates in the Fb-CM group and the SO-CM group were significantly higher than that in the SOM group (P<0.05). After 48 hours of culture, the level of reactive oxygen species in the SO-CM group was significantly higher than that in the SOM group and the Fb-CM group (with both P values <0.05). After 48 hours of culture, there was no statistically significant difference in the rate of β-galactosidase-positive staining of cells among the three groups (P>0.05). Conclusions The SO-CM has high content of VEGF and can significantly promote the proliferation, migration, and expression of reactive oxygen species in human dermal Fbs induced by high glucose, but has no significant effect on cell senescence. -
Key words:
- Culture media, conditioned /
- Fibroblasts /
- Cell proliferation /
- Reactive oxygen species /
- Cell aging /
- Cell migration /
- Skin organoid
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刘志欣、仇恺真：设计并实施研究、采集和分析数据；何佳、王婧薷、刘必来：论文修改、实验指导；信琪、李桂强：实施研究；陈晓东：设计实验、论文撰写、论文评阅、资金支持所有作者声明不存在利益冲突
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参考文献
(41) [1] WilkinsonHN,HardmanMJ.Wound healing: cellular mechanisms and pathological outcomes[J].Open Biol,2020,10(9):200223.DOI: 10.1098/rsob.200223. [2] HollJ,KowalewskiC,ZimekZ,et al.Chronic diabetic wounds and their treatment with skin substitutes[J].Cells,2021,10(3):655.DOI: 10.3390/cells10030655. [3] desJardins-ParkHE,FosterDS,LongakerMT.Fibroblasts and wound healing: an update[J].Regen Med,2018,13(5):491-495.DOI: 10.2217/rme-2018-0073. [4] TalbottHE,MascharakS,GriffinM,et al.Wound healing, fibroblast heterogeneity, and fibrosis[J].Cell Stem Cell,2022,29(8):1161-1180.DOI: 10.1016/j.stem.2022.07.006. [5] Al-RikabiAHA, TobinDJ, Riches-SumanK,et al.Dermal fibroblasts cultured from donors with type 2 diabetes mellitus retain an epigenetic memory associated with poor wound healing responses[J].Sci Rep,2021,11(1):1474.DOI: 10.1038/s41598-020-80072-z. [6] LiuY,LiuY,HeW,et al.Fibroblasts: immunomodulatory factors in refractory diabetic wound healing[J].Front Immunol,2022,13:918223.DOI: 10.3389/fimmu.2022.918223. [7] RaiV,MoellmerR,AgrawalDK.Role of fibroblast plasticity and heterogeneity in modulating angiogenesis and healing in the diabetic foot ulcer[J].Mol Biol Rep,2023,50(2):1913-1929.DOI: 10.1007/s11033-022-08107-4. [8] DashBC,KorutlaL,VallabhajosyulaP,et al.Unlocking the potential of induced pluripotent stem cells for wound healing: the next frontier of regenerative medicine[J].Adv Wound Care (New Rochelle),2022,11(11):622-638.DOI: 10.1089/wound.2021.0049. [9] ShafieeA,SunJ,AhmedIA,et al.Development of physiologically relevant skin organoids from human induced pluripotent stem cells[J].Small,2024,20(16):e2304879.DOI: 10.1002/smll.202304879. [10] YiSA,ZhangY,RathnamC,et al.Bioengineering approaches for the advanced organoid research[J].Adv Mater,2021,33(45):e2007949.DOI: 10.1002/adma.202007949. [11] HoangP,MaZ.Biomaterial-guided stem cell organoid engineering for modeling development and diseases[J].Acta Biomater,2021,132:23-36.DOI: 10.1016/j.actbio.2021.01.026. [12] LeeJ,RabbaniCC,GaoH,et al.Hair-bearing human skin generated entirely from pluripotent stem cells[J].Nature,2020,582(7812):399-404.DOI: 10.1038/s41586-020-2352-3. [13] DaneshmandiL,ShahS,JafariT,et al.Emergence of the stem cell secretome in regenerative engineering[J].Trends Biotechnol,2020,38(12):1373-1384.DOI: 10.1016/j.tibtech.2020.04.013. [14] L PK,KandoiS,MisraR,et al.The mesenchymal stem cell secretome: a new paradigm towards cell-free therapeutic mode in regenerative medicine[J].Cytokine Growth Factor Rev,2019,46:1-9.DOI: 10.1016/j.cytogfr.2019.04.002. [15] BormannD,GugerellA,AnkersmitHJ,et al.Therapeutic application of cell secretomes in cutaneous wound healing[J].J Invest Dermatol,2023,143(6):893-912.DOI: 10.1016/j.jid.2023.02.019. [16] QianJ,LuE,XiangH,et al.GelMA loaded with exosomes from human minor salivary gland organoids enhances wound healing by inducing macrophage polarization[J].J Nanobiotechnology,2024,22(1):550.DOI: 10.1186/s12951-024-02811-y. [17] KwakS,SongCL,LeeJ,et al.Development of pluripotent stem cell-derived epidermal organoids that generate effective extracellular vesicles in skin regeneration[J].Biomaterials,2024,307:122522.DOI: 10.1016/j.biomaterials.2024.122522. [18] 边晓玮,张翠萍,李炳旻,等.胎盘间充质干细胞外泌体对高糖培养的成纤维细胞衰老的影响[J].第三军医大学学报,2020,42(12):1163-1173.DOI: 10.16016/j.1000-5404.202001190. [19] GantwerkerEA,HomDB.Skin: histology and physiology of wound healing[J].Facial Plast Surg Clin North Am,2011,19(3):441-453.DOI: 10.1016/j.fsc.2011.06.009. [20] ParkJW,HwangSR,YoonIS.Advanced growth factor delivery systems in wound management and skin regeneration[J].Molecules,2017,22(8):1259.DOI: 10.3390/molecules22081259. [21] XueM,ZhaoR,MarchL,et al.Dermal fibroblast heterogeneity and its contribution to the skin repair and regeneration[J].Adv Wound Care (New Rochelle),2022,11(2):87-107.DOI: 10.1089/wound.2020.1287. [22] LermanOZ,GalianoRD,ArmourM,et al.Cellular dysfunction in the diabetic fibroblast: impairment in migration, vascular endothelial growth factor production, and response to hypoxia[J].Am J Pathol,2003,162(1):303-312.DOI: 10.1016/S0002-9440(10)63821-7. [23] ZhangP,SongX,DongQ,et al.miR-27-3p inhibition restore fibroblasts viability in diabetic wound by targeting NOVA1[J].Aging (Albany NY),2020,12(13):12841-12849.DOI: 10.18632/aging.103266. [24] WangZ,ZhaoF,LangH,et al.Organoids in skin wound healing[J/OL].Burns Trauma,2025,13:tkae077[2025-02-09].http://www.ncbi.nlm.nih.gov/pubmed/39759541.DOI: 10.1093/burnst/tkae077. [25] WangW,LiuP,ZhuW,et al.Skin organoid transplantation promotes tissue repair with scarless in frostbite[J/OL].Protein Cell,2024:pwae055(2024-10-04)[2024-10-20]. https://pubmed.ncbi.nlm.nih.gov/39363875/.DOI:10.1093/procel/pwae055.[published online ahead of print]. [26] BaoP,KodraA,Tomic-CanicM,et al.The role of vascular endothelial growth factor in wound healing[J].J Surg Res,2009,153(2):347-358.DOI: 10.1016/j.jss.2008.04.023. [27] GoswamiAG,BasuS,HudaF,et al.An appraisal of vascular endothelial growth factor (VEGF): the dynamic molecule of wound healing and its current clinical applications[J].Growth Factors,2022,40(3/4):73-88.DOI: 10.1080/08977194.2022.2074843. [28] ShamsF,MoravvejH,HosseinzadehS,et al.Overexpression of VEGF in dermal fibroblast cells accelerates the angiogenesis and wound healing function: in vitro and in vivo studies[J].Sci Rep,2022,12(1):18529.DOI: 10.1038/s41598-022-23304-8. [29] RoyR,ZayasJ,MohamedMF,et al.IL-10 dysregulation underlies chemokine insufficiency, delayed macrophage response, and impaired healing in diabetic wounds[J].J Invest Dermatol,2022,142(3Pt A):692-704.e14.DOI: 10.1016/j.jid.2021.08.428. [30] SaraivaM,O'GarraA.The regulation of IL-10 production by immune cells[J].Nat Rev Immunol,2010,10(3):170-181.DOI: 10.1038/nri2711. [31] IshidaY,KuninakaY,NosakaM,et al.CCL2-mediated reversal of impaired skin wound healing in diabetic mice by normalization of neovascularization and collagen accumulation[J].J Invest Dermatol,2019,139(12):2517-2527.e5.DOI: 10.1016/j.jid.2019.05.022. [32] WoodS,JayaramanV,HuelsmannEJ,et al.Pro-inflammatory chemokine CCL2 (MCP-1) promotes healing in diabetic wounds by restoring the macrophage response[J].PLoS One,2014,9(3):e91574.DOI: 10.1371/journal.pone.0091574. [33] OhM,KimYJ,SonYJ,et al. Promotive effects of human induced pluripotent stem cell-conditioned medium on the proliferation and migration of dermal fibroblasts[J]. Biotechnol Bioproc E, 2017, 22 (5): 561-568. DOI: 10.1007/s12257-017-0221-1. [34] YanY,WuR,BoY,et al.Induced pluripotent stem cells-derived microvesicles accelerate deep second-degree burn wound healing in mice through miR-16-5p-mediated promotion of keratinocytes migration[J].Theranostics,2020,10(22):9970-9983.DOI: 10.7150/thno.46639. [35] OhM,LeeJ,KimYJ,et al.Exosomes derived from human induced pluripotent stem cells ameliorate the aging of skin fibroblasts[J].Int J Mol Sci,2018,19(6):1715.DOI: 10.3390/ijms19061715. [36] ZhangJ,GuanJ,NiuX,et al.Exosomes released from human induced pluripotent stem cells-derived MSCs facilitate cutaneous wound healing by promoting collagen synthesis and angiogenesis[J].J Transl Med,2015,13:49.DOI: 10.1186/s12967-015-0417-0. [37] ZhouAK,JouE,LuV,et al.Using pre-clinical studies to explore the potential clinical uses of exosomes secreted from induced pluripotent stem cell-derived mesenchymal stem cells[J].Tissue Eng Regen Med,2023,20(6):793-809.DOI: 10.1007/s13770-023-00557-6. [38] KobayashiH,EbisawaK,KambeM,et al.<Editors' Choice> Effects of exosomes derived from the induced pluripotent stem cells on skin wound healing[J].Nagoya J Med Sci,2018,80(2):141-153.DOI: 10.18999/nagjms.80.2.141. [39] KimS,LeeSK,KimH,et al.Exosomes secreted from induced pluripotent stem cell-derived mesenchymal stem cells accelerate skin cell proliferation[J].Int J Mol Sci,2018,19(10):3119.DOI: 10.3390/ijms19103119. [40] DunnillC,PattonT,BrennanJ,et al.Reactive oxygen species (ROS) and wound healing: the functional role of ROS and emerging ROS-modulating technologies for augmentation of the healing process[J].Int Wound J,2017,14(1):89-96.DOI: 10.1111/iwj.12557. [41] HuoYN,ChenW,ZhengXX.ROS, MAPK/ERK and PKC play distinct roles in EGF-stimulated human corneal cell proliferation and migration[J].Cell Mol Biol (Noisy-le-grand),2015,61(7):6-11. -
图 1 诱导培养人iPSC来源的皮肤类器官的各时间点的iPSC聚集体形态。1A、1B、1C.分别为诱导培养第0（加入促进分化的Essential 6培养基后即刻）、3、6天,图1A可见iPSC聚集体开始分化,图1B和图1C可见iPSC聚集体出现胚层结构 光学显微镜×100；1D、1E、1F.分别为诱导培养第33、57、110天,图1D可见iPSC聚集体出现明显的头尾结构,图1E可见iPSC聚集体出现毛囊的初级结构,图1F可见皮肤类器官上有明显的毛囊结构 光学显微镜×40
注：iPSC为诱导多能干细胞；黄色箭头指示尾结构,红色箭头指示头结构,绿色箭头指示毛囊初级结构,蓝色箭头指示毛囊结构
图 2 3组高糖诱导的人真皮成纤维细胞培养24 h后的Ki67表达情况 花青素3-4',6-二脒基-2-苯基吲哚×100。2A、2B、2C.分别为SOM组细胞核染色、Ki67阳性染色、细胞核与Ki67阳性染色重叠图片；2D、2E、2F.分别为Fb-CM组细胞核染色、Ki67阳性染色、细胞核与Ki67阳性染色重叠图片,图2F中Ki67阳性表达明显高于图2C；2G、2H、2I.分别为SO-CM组细胞核染色、Ki67阳性染色、细胞核与Ki67阳性染色重叠图片,图2I中Ki67阳性表达明显高于图2C
注：皮肤类器官培养基（SOM）组、成纤维细胞条件培养基（Fb-CM）组、皮肤类器官条件培养基（SO-CM）组分别使用均含终物质的量浓度35 mmol/L葡萄糖的SOM、Fb-CM、SO-CM培养细胞；细胞核阳性染色为蓝色；Ki67阳性染色为红色,表示细胞增殖活力
图 3 3组高糖诱导的人真皮成纤维细胞划痕后各时间点迁移情况 倒置荧光显微镜×100。3A、3B、3C.分别为SOM组、Fb-CM组、SO-SM组划痕后0 h（即刻）的划痕面积,3组划痕面积相近；3D、3E、3F.分别为SOM组、Fb-CM组、SO-SM组划痕后13 h的划痕面积,其中图3D剩余的划痕面积明显大于图3E和图3F
注：皮肤类器官培养基（SOM）组、成纤维细胞条件培养基（Fb-CM）组、皮肤类器官条件培养基（SO-CM）组分别使用均含终物质的量浓度35 mmol/L葡萄糖的SOM、Fb-CM、SO-CM培养细胞
图 4 3组高糖诱导的人真皮成纤维细胞培养48 h后活性氧表达水平 2',7'-二氯二氢荧光素二乙酸酯×200。4A、4B、4C.分别为SOM组、Fb-CM组、SO-CM组的活性氧表达,图4C活性氧表达水平明显高于图4A和图4B
注：皮肤类器官培养基（SOM）组、成纤维细胞条件培养基（Fb-CM）组、皮肤类器官条件培养基（SO-CM）组分别使用均含终物质的量浓度35 mmol/L葡萄糖的SOM、Fb-CM、SO-CM培养细胞；活性氧阳性染色为绿色
图 5 3组高糖诱导的人真皮成纤维细胞培养48 h后β-半乳糖苷酶表达情况 倒置荧光显微镜×200。5A、5B、5C.分别为SOM组、Fb-CM组、SO-CM组β-半乳糖苷酶染色,3组无明显差别
注：皮肤类器官培养基（SOM）组、成纤维细胞条件培养基（Fb-CM）组、皮肤类器官条件培养基（SO-CM）组分别使用均含终物质的量浓度35 mmol/L葡萄糖的SOM、Fb-CM、SO-CM培养细胞；衰老细胞的细胞核周围呈蓝绿色,未衰老细胞不着色
Table 1. 3种培养基中细胞因子含量比较（pg/mL,
培养基类型 样本数 TNF-α IL-10 IL-18 CCL-2 VEGF VEGF-β PDGF TGF-β EGF SOM 3 0.39±0.68 2.63±0.94 4.5±3.7 1.46±0.26 1.0±0.5 1.41±0.03 2.80±0.28 5.0±0.5 2.26±0.27 Fb-CM 3 0.00±0.00 0.34±0.30 1.9±2.0 206.80±14.80 106.5±36.9 0.07±0.12 6.16±3.64 9.7±6.0 0.74±0.30 SO-CM 3 0.62±1.08 0.31±0.27 10.2±3.4 154.50±24.24 339.2±54.2 0.07±0.06 2.92±1.15 5.6±0.4 0.65±0.27 统计量值 H=0.13 F=15.14 H=4.86 H=7.20 H=7.20 H=5.66 H=4.36 H=5.07 F=31.11 P值 0.999 0.005 0.082 0.004 0.004 0.061 0.132 0.086 <0.001 P1值 0.999 0.008 0.999 0.022 0.539 0.099 0.303 0.076 0.001 P2值 0.999 0.008 0.465 0.539 0.022 0.143 0.999 0.999 0.001 P3值 0.999 0.998 0.090 0.539 0.539 0.999 0.158 0.539 0.921 注：SOM为皮肤类器官培养基,Fb-CM为成纤维细胞条件培养基,SO-CM为皮肤类器官条件培养基,TNF-α为肿瘤坏死因子α,IL为白细胞介素,CCL-2为CC类趋化因子配体2,VEGF为血管内皮生长因子,PDGF为血小板源性生长因子,TGF-β为转化生长因子β,EGF为表皮生长因子；F值、P值为各类型培养基间各指标总体比较所得,P1值、P2值、P3值分别为SOM与Fb-CM、SOM与SO-CM、Fb-CM与SO-CM各指标比较所得 
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Table 2. 3组高糖诱导的人真皮成纤维细胞培养24 h后的细胞增殖活力比较（
组别 样本数 Ki67阳性细胞比（%） 吸光度值 SOM组 3 29.6±2.1 100±6 Fb-CM组 3 45.2±6.0 124±5 SO-CM组 3 57.4±4.0 158±12 F值 30.99 34.31 P值 <0.001 <0.001 P1值 0.011 0.031 P2值 <0.001 <0.001 P3值 0.032 0.007 注：皮肤类器官培养基（SOM）组、成纤维细胞条件培养基（Fb-CM）组、皮肤类器官条件培养基（SO-CM）组分别使用均含终物质的量浓度35 mmol/L葡萄糖的SOM、Fb-CM、SO-CM培养细胞；F值、P值为组间各指标总体比较所得,P1值、P2值、P3值分别为SOM组与Fb-CM组、SOM组与SO-CM组、Fb-CM组与SO-CM组各指标比较所得
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